WO2000064485A2 - Specifically targeted catalytic antagonists and uses thereof - Google Patents
Specifically targeted catalytic antagonists and uses thereof Download PDFInfo
- Publication number
- WO2000064485A2 WO2000064485A2 PCT/US2000/010988 US0010988W WO0064485A2 WO 2000064485 A2 WO2000064485 A2 WO 2000064485A2 US 0010988 W US0010988 W US 0010988W WO 0064485 A2 WO0064485 A2 WO 0064485A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- enzyme
- residue
- cysteine
- targeting moiety
- antagonist
- Prior art date
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Classifications
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Definitions
- This invention relates to the field of chimeric molecules.
- this invention provides novel chimeric molecules that act as catalytic antagonists of targets (e.g. receptors, enzymes, lectins, etc.).
- targets e.g. receptors, enzymes, lectins, etc.
- a chimeric molecule two or more molecules that exist separately in their native state are joined together to form a single molecule having the desired functionality of all of its constituent molecules.
- one of the constituent molecules of a chimeric molecule is a "targeting molecule”.
- the targeting molecule is a molecule such as an antibody that specifically binds to its corresponding target and, by virtue of the targeting molecule, the chimeric molecule will specifically bind (target) cells and tissues bearing the target (e.g. the epitope) to which the targeting moiety is directed.
- the effector molecule refers to a molecule that is to be specifically transported to the target to which the chimeric molecule is specifically directed.
- Chimeric molecules comprising a targeting moiety attached to an effector moiety have been used in a wide variety of contexts. Thus, for example, chimeric molecules comprising a targeting moiety joined to a cytotoxic "effector molecule” have frequently been used to target and kill tumor cells (see, e.g., Pastan et al, Ann. Rev. Biochem., 61: 331-354 (1992).
- Other chimeric molecules comprising a targeting moiety attached to angiogenesis inhibitors have been used to inhibit tumor growth and/or proliferation.
- angiogenesis inducers have been proposed for the treatment of atherosclerosis.
- Other uses of chimeric molecules have involved the delivery of intrabodies, intracellularly expressed antibodies that then bind to an intracellular protein, the specific delivery of vectors (e.g. for gene therapy), or the creation of tissue-specific liposomes.
- the target recognized by the targeting moiety is not the desired site of action of the effector molecule.
- chimeric cytotoxins used to treat cancers e.g. IL4-PE, BlFvPE38, etc., see, e.g., Benhar & Pastan (1995) Clin. Cane. Res., 1: 1023-1029, Thrush et al. (1996) Ann. Rev.
- the targeting moiety specifically binds to a target on the surface of the cell.
- the chimeric molecule is then internalized into the cell and the effector molecule (e.g., ricin, abrin, Diptheria toxin, Pseudomonas exotoxin) is transported to the cytosol of the cell where it exerts its characteristic activity (e.g. ADP ribosylation in the case o ⁇ Pseudomonas exotoxin).
- the effector molecule e.g., ricin, abrin, Diptheria toxin, Pseudomonas exotoxin
- targeted liposomes are typically internalized through a receptor- mediated process or through the action of the lipid.
- Targeted intrabodies and gene therapy vectors are also internalized for expression within the cell.
- a common goal in the design of targeted chimeric molecules has been the increase of binding specificity and avidity. It is generally believed that, by increasing avidity and specificity the concentration of the chimeric molecule to achieve a given result will decrease. Thus, release of the chimeric molecule from its target is generally viewed as undesirable.
- chimeric molecules Because the chimeric molecule is typically internalized (in the case of targeted cells) and the activity of the effector molecule is directed to a molecule other than the specifically recognized target, chimeric molecules typically act in a "stoichiometric" manner. That is, each chimeric molecule is essentially consumed upon interaction with its "substrate” and activity of the chimeric molecule is unavailable for subsequent reactions. As a consequence chimeric molecules must be maintained at relatively high level for efficacy and a recurring problem of chimeric moieties, particularly in in vivo applications is the inability to maintain elevated serum levels of the chimeric molecule over therapeutically significant periods of time and the increased (e.g. non-specific) toxicity caused by the high dosages that must be utilized.
- the molecules of this invention specifically bind to a target molecule and degrade that bound molecule. In preferred embodiments, this results in a loss of activity (e.g. biological activity) of the target molecule and also results in the release of the chimeric molecule so that it is free to find and degrade another target. In this manner the chimeric molecule is "regenerated” and essentially catalytic. Because a single chimeric molecule can attack and degrade an essentially limitless number of targets, the so called “catalytic antagonists" of this invention are highly effective at relatively low dosages.
- this invention provides a catalytic antagonist of a target molecule (e.g. an enzyme, a receptor, etc.).
- the antagonist comprises a targeting moiety that specifically binds to the target molecule and the targeting moiety is attached to an enzyme that degrades the target molecule to reduce binding of the target molecule to its cognate ligand.
- the degradation of the target molecule also reduces binding of the antagonist to the target molecule.
- the antagonist is released from the target thereby allowing the antagonist to bind and degrade another target molecule.
- the targeting moiety is joined to the enzyme through the sulfur group on a cysteine and the cysteine is a naturally occurring cysteine in the enzyme or a cysteine introduced into the enzyme (e.g. substituted for a native amino acid other than cysteine in the enzyme).
- the cysteine is a cysteine that is substituted for a native amino acid other than cysteine in or near a subsite comprising a substrate binding site of the enzyme.
- the cysteine is a cysteine that is substituted for an amino acid forming a substrate binding site.
- Preferred enzymes include, but are not limited to a protease, an esterase, an amidase, a peptidase, a lactamase, a cellulase, an oxidase, an oxidoreductase, a reductase, a transferase, a hydrolase, an isomerase, a ligase, a lipase, a phospholipase, a phosphatase, a kinase, a sulfatase, a lysozyme, a glycosidase, a nuclease, an aldolase, a ketolase, a lyase, a cyclase, a reverse transcriptase, a hyaluronidase, an amylase, a cerebrosidase, and a chitinase.
- the enzyme is a serine hydrolase.
- the enzyme is a subtilisin-type serine hydrolase (e.g. a Bacillus lentus subtilisin) and said cysteine is substituted for an amino acid in or near a subsite selected from the group consisting of an SI subsite, an SI' subsite, and an S2 subsite.
- the enzyme is a Bacillus lentus subtilisin.
- the cysteine is substituted for an amino acid in a subtillisin, where the amino acid corresponds to a reference residue in a Bacillus lentus subtilisin, where the reference residue is at or near a residue selected from the group consisting of residue 156, residue 166, residue 217, residue 222, residue 62, residue 96, residue 104, residue 107, residue 189, and residue 209.
- the enzyme is a chymotrypsin-type serine protease and the cysteine is substituted for the amino acid corresponding to a reference residue in a mature trypsin (Protein Data Bank entry 1TPP), wherein said reference residue is at or near a residue selected from the group consisting of Tyr94, Leu99, Glnl75, Aspl89, Serl90, Glnl92, Phe41, Lys60, Tyrl51, Ser214, and Lys224.
- a reference residue selected from the group consisting of Tyr94, Leu99, Glnl75, Aspl89, Serl90, Glnl92, Phe41, Lys60, Tyrl51, Ser214, and Lys224.
- the enzyme is an alpha/beta type serine hydrolase and the cysteine is substituted for the amino acid corresponding to a reference residue in a Candida antartica lipase (Protein Data Bank entry 1TCA), where the reference residue is at or near a residue selected from the group consisting of Trpl04, Leu 140, Leu 144, Val 154, Glul88, Ala 225, Leu278 and Ile285.
- the enzyme is an aspartyl protease. More preferably the enzyme is a pepsin-type protease and the cysteine is substituted for the amino acid corresponding to a reference residue in the mature human pepsin (Protein Data Bank entry 1PSN), where the reference residue is at or near a residue selected from the group consisting of Tyr9, Metl2, Glul3, Gly76, Thr77, Phel l l, Phel l7, Ilel28, Serl30, Tyrl89, Ile213, Glu239, Met245, Gln287, Met289, Leu291, and Glu294.
- the enzyme is a cysteine protease.
- the enzyme is a papain and the cysteine is substituted for the amino acid corresponding to a reference residue in a mature papain (Protein Data Bank entry 1BQI), where the reference residue is at or near a residue selected from the group consisting of Asnl ⁇ , Ser21, Asn64, Tyr67, Trp69, Glnl 12, Gin 142, Aspl58, Trpl77, and Phe207.
- the enzyme is a metalloprotease and the cysteine is substituted for the amino acid corresponding to a reference residue in the mature human matrix metalloprotease (Protein Data Bank entry 830C), where the reference residue is at or near a residue selected from the group consisting of Leul 11, Phel75, Tyrl76, Serl82, Leul84, Phel89, Tyr214, Asp231, Lys234, and Ile243.
- the catalytic antagonist targeting moiety is directed against a target where the target is a molecule present on the surface of a cell (e.g., a molecule forming a receptor, a ligand, a component of a cell wall, a component of a cell membrane, etc.).
- the targeting moiety includes, but is not limited to an antigen , a carbohydrate, a nucleic acid, a lipid, a coordination complex, a sugar, a vitamin, a dendrimer, and a crown ether.
- the targeting moiety is a cognate ligand for a receptor or an enzyme.
- the targeting moiety is an inhibitor for a receptor or an enzyme.
- the enzyme is a protease (e.g. a papain, a subtilisin, a pepsin, a trypsin, a metalloprotease, etc.) and the targeting moiety is a ligand selected from the group consisting of a carbohydrate, a vitamin or vitamin analog, an enzyme inhibitor, a peptide, a pharmaceutical that is a small organic molecule, and biotin.
- the enzyme is a protease and said targeting moiety is a receptor.
- the enzyme is a protease (e.g. a papain, a subtilisin, a pepsin, a trypsin, a metalloprotease, etc.) and the targeting moiety is an enzyme inhibitor that is a pyrazole, a biotin, a ligand that binds a lectin (e.g. concanavalin A), a carbohydrate (e.g. thioethyl D-mannopyranoside).
- the targeting moiety specifically binds to a soil and the enzyme degrades a component of the soil.
- this invention provides a method of degrading a target molecule.
- the method involves contacting the target molecule with a catalytic antagonist comprising a targeting moiety that specifically binds to the target molecule the targeting moiety being attached to an enzyme that degrades the target molecule.
- the degradation of the target molecule releases the antagonist thereby allowing the antagonist to bind and degrade another target molecule.
- the targeting moiety is joined to the enzyme through the sulfur group on a cysteine.
- Preferred antagonist molecules include, but are not limited to the catalytic antagonist molecules described above.
- this invention provides an enzyme having altered substrate specificity (i.e. a "redirected enzyme).
- the enzyme preferably comprises a targeting moiety attached to a subsite comprising the substrate binding site of said enzyme.
- the targeting moiety is coupled to said enzyme through to a sulfur of a cysteine in said subsite of said enzyme.
- the cysteine may be a native cysteine or a cysteine is substituted for a native amino acid that is not cysteine in the subsite of the enzyme.
- Preferred enzymes include, but are not limited to a protease, an esterase, an amidase, a peptidase, a lactamase, a cellulase, an oxidase, an oxidoreductase, a reductase, a transferase, a hydrolase, an isomerase, a ligase, a lipase, a phospholipase, a phosphatase, a kinase, a sulfatase, a lysozyme, a glycosidase, a glycosyltransferase, a nuclease, an aldolase, a ketolase, a lyase, a cyclase, a reverse transcriptase, a hyaluronidase, an amylase, a cerebrosidase and a chitinase.
- the enzyme is a serine hydrolase (e.g., a subtilisin).
- the cysteine is preferably subsitited for amino acids at or near a subsite selected from the group consisting of an SI subsite, an ST subsite, and an S2 subsite.
- Particularly preferred sites for substitution of the cysteine in various enzymes include, but are not limited to those identified above.
- particularly preferred targets and targeting moieties include those identified above.
- the targeting moiety is an inhibitor for a receptor or an enzyme, in other embodiments the targeting moiety is selected from the group consisting of a growth factor, a cytokine, and a receptor ligand.
- the enzyme is a protease and the targeting moiety is a ligand selected from the group consisting of a carbohydrate, a vitamin or vitamin analog, an enzyme inhibitor, a peptide, a pharmaceutical that is a small organic molecule, and biotin.
- the enzyme is a protease (e.g. a subtilisin, a papain, a pepsin, etc.) and the targeting moiety is a receptor, enzyme inhibitor that is a pyrazole, a biotin, a ligand that binds a lectin (e.g. concanavalin A), or a carbohydrate (e.g. thioethyl D- mannopyranoside).
- the targeting moiety specifically binds to a soil and said enzyme degrades a component of the soil.
- this invention provides methods of directing the activity of an enzyme to a specific target.
- the methods comprise providing an enzyme having altered substrate specificity said enzyme comprising a targeting moiety attached to a subsite within the substrate binding region of said enzyme; and contacting the target with the enzyme, whereby the enzyme specifically binds to the target thereby localizing the activity of the enzyme at the target.
- Preferred enzymes include, but are not limited to, the "redirected" enzymes described above.
- This invention also provides methods of enhancing the activity of a drug that acts as an inhibitor of a receptor or an enzyme. The methods involve coupling a hydrolase to said drug such that when said drug binds said receptor or enzyme, the hydrolase degrades the receptor or enzyme. In preferred embodiments, the method increases the dosage therapeutic window of said drug.
- the hydrolase is a serine hydrolase (e.g. a subtilisin).
- the hydrolase is a metalloprotease, a cysteine protease, an aspartyl protease, and the like.
- This invention also provides a method of inhibiting an enzyme or a receptor.
- the method comprises contacting the enzyme or receptor with a chimeric molecule comprising a ligand that binds the enzyme or receptor attached to an enzyme that degrades the cognate ligand of the enzyme or receptor.
- the enzyme thus becomes linked to the enzyme or receptor where it is free to degrade the cognate ligand thereby preventing the cognate ligand from activating the receptor or acting as a substrate for the enzyme.
- the chimeric molecule comprises a hydrolase (e.g. a protease) attached to an inhibitor of the enzyme or receptor.
- Preferred hydrolases include, but are not limited to a serine protease, a cysteine protease, an aspartyl protease, a pepsin-type protease, and a metalloprotease.
- this invention does not include catalytic antibodies, e.g. as described by Hifumi et al. (1999) J. Bioscience and Bioengineering, 88: 323.
- catalytic antagonist refers to an enzyme that can inhibit the activity of a molecule that has a particular biological activity and/or simply degrade a molecule that has no particular biological activity.
- the inhibition can be a blocking or destroying of the function of the "target” molecule.
- the inhibition or blockage is by partial or complete degradation of the target molecule.
- the “catalytic antagonist” is catalytic by virtue of the fact that the antagonist is not itself consumed or significantly altered (i.e., permanently changed) by its interaction with the target molecule.
- the degradation of the target molecule ultimately results in the release of the catalytic antagonist so that it is free to attack another target molecule.
- the reaction is preferably sub-stoichiometric (ratio of catalytic antagonist to target is less than 1) and a single catalytic antagonist is free to degrade any number of target molecules.
- a "target molecule” refers to a molecule that is specifically bound by the catalytic antagonist or specifically directed enzymes described herein. Where a catalytic antagonist is employed the target molecule is partially or completely degraded by that antagonist.
- a “targeting moiety” refers to a moiety in the chimeric molecule that that specifically binds to the target molecule. Prior to coupling the targeting moiety to the enzyme, the targeting moiety is a targeting molecule. In preferred embodiments, the targeting moiety is one of a pair of cognate binding partners.
- a heterogeneous population of molecules e.g., proteins and other biologies.
- the binding may be by one or more of a variety of mechanisms including, but not limited to ionic interactions, covalent interactions, hydrophobic interactions, van der Waals interactions, etc.
- binding partner or a member of a “binding pair”, or “cognate ligand” refers to molecules that specifically bind other molecules to form a binding complex such as antibody/antigen, lectin/carbohydrate, nucleic acid/nucleic acid, receptor/receptor ligand (e.g. IL-4 receptor and IL-4), avidin/biotin, etc.
- ligand is used to refer to a molecule that specifically binds to another molecule. Commonly a ligand is a soluble molecule, e.g. a hormone or cytokine, that binds to a receptor.
- amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
- the term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide.
- Proteins also include glycoproteins (e.g. histidine-rich glycoprotein (HRG), Lewis Y antigen (Le ⁇ ), and the like.).
- nucleic acid or “oligonucleotide” or grammatical equivalents herein refer to at least two nucleotides covalently linked together.
- a nucleic acid of the present invention is preferably single-stranded or double stranded and will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10): 1925) and references therein; Letsinger (1970) J. Org. Chem. 35:3800; Sblul et al. (1977) Eur. J. Biochem.
- nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al. (1995), Chem. Soc. Rev. pp 169- 176).
- nucleic acid analogs are described in Rawls, C & E News June 2, 1997 page 35. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.
- the term "residue” as used herein refers to natural, synthetic, or modified amino acids.
- enzyme includes proteins that are capable of catalyzing chemical changes in other substances without being permanently changed themselves.
- the enzymes can be wild-type enzymes or variant enzymes.
- Enzymes within the scope of the present invention include, but are not limited to, proteases, esterases, amidases, peptidases, lactamases, cellulases, oxidases, oxidoreductases, reductases, transferases, hydrolases, isomerases, ligases, lipases, phospholipases, phosphatases, kinases, sulfatases, lysozymes, glycosidases, glycosyltransferases, nucleases, aldolases, ketolases, lyases, cyclases, reverse transcriptases, hyaluronidases, amylases, cerebrosidases, chitinases, and the like.
- a "mutant enzyme” is an enzyme that has been changed by replacing an amino acid
- a “chemically modified” enzyme is an enzyme that has been derivatized to bear a substituent not normally found at that location in the enzyme.
- the derivatization typically is of a post translational modification, occasionally performed in vivo, but more typically performed ex vivo.
- a “chemically modified mutant enzyme” or “CMM” is an enzyme in which an amino acid residue has been replaced with another amino acid residue (preferably a cysteine) and the replacement residue is chemically derivatized to bear a substituent not normally found on that residue.
- the term "thiol side chain group”, “thiol containing group”, and “thiol side chain” are terms that can be used interchangeably and include groups that are used to replace the thiol hydrogen of a cysteine. Commonly the thiol side chain group includes a sulfur atom through which the thiol side chain group that is attached to the thiol sulfur of the cysteine.
- substituted typically refers to the group remains attached to the cysteine through a disulfide linkage formed by reacting the cysteine with a methanesulfonate reagent as described herein. While the term substituent preferably refers just to the group that remains attached (excluding its thiol group), the substituent can also refer to the entire thiol side chain group. The difference will be clear from the context.
- the "binding site of an enzyme” consists of a series of subsites across the substrate binding surface of the enzyme (Berger & Schechter (1970) Phil. Trans. Roy Soc. Lond. B 257: 249-264).
- the substrate residues that correspond to the subsites are labeled P and the subsites are labeled S.
- the subsites are labeled Si, S , S 3 , S 4 , Si', and S 2 '.
- a discussion of subsites can be found in Siezen et al. (1991) Protein Engineering, 4: 719-737, and Fersht (1985) Enzyme Structure and Mechanism, 2nd ed. Freeman, New York, 29-30.
- the preferred subsites include Si, Si', and S 2 .
- the phrase " amino acid ##" or “amino acid ## in the XX subsite” is intended to include the amino acid at the referenced position (e.g. amino acid 156 of 5. lentus subtilisin which is in the Si subsite) and the amino acids at the corresponding (homologous) position in related enzymes.
- a residue (amino acid) of an enzyme is equivalent to a residue of a referenced enzyme (e.g. B. amyloliquefaciens subtilisin) if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in B. amyloliquefaciens subtilisin (i.e., having the same or similar functional capacity to combine, react, or interact chemically).
- a referenced enzyme e.g. B. amyloliquefaciens subtilisin
- the amino acid sequence of the subject enzyme e.g. a serine hydrolase, cysteine protease, aspartyl protease, metalloprotease, etc.
- a reference enzyme e.g. B. amyloliquefaciens subtilisin in the case of a subtilisin type serine protease
- a set of residues known to be invariant in all enzymes of that family e.g. subtilisins
- the residues equivalent to particular amino acids in the primary sequence of the reference enzyme e.g. B. amyloliquefaciens subtilisin
- Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of greater than 75% or as little as 50% of conserved residues is also adequate to define equivalent residues.
- Conservation of the catalytic triad, e.g., Asp32/His64/Ser221) should be maintained for serine hydrolases.
- the conserved residues may be used to define the corresponding equivalent amino acid residues in other related enzymes.
- the two (reference and "target") sequences are aligned in order to produce the maximum homology of conserved residues.
- a number of deletions are seen in the thermitase sequence as compared to B. amyloliquefaciens subtilisin (see, e.g. U.S. Patent 5,972,682).
- the equivalent amino acid of Tyr217 in B. amyloliquefaciens subtilisin in thermitase is the particular lysine shown beneath Tyr217 in Figure 5B-2 of the 5,972,682 patent.
- Equivalent resides may be substituted by a different amino acid to produce a mutant carbonyl hydrolase since they are equivalent in primary structure.
- Equivalent residues homologous at the level of tertiary structure for a particular enzyme whose tertiary structure has been determined by x-ray crystallography are defined as those for which the atomic coordinates of 2 or more of the main chain atoms of a particular amino acid residue of the reference sequence (e.g. B. amyloliquefaciens subtilisin) and the sequence in question (target sequence) (N on N, CA on CA, C on C, and O on O) are within 0J3 nm and preferably 0J nm after alignment.
- Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the enzyme in question to the reference sequence.
- the best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.
- Equivalent residues which are functionally analogous to a specific residue of a reference sequence are defined as those amino acids sequence in question (e.g. related subtilisin) which may adopt a conformation such that they will alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the reference sequence as described herein.
- a “reference residue” refers to a residue that is specified in a particular enzyme and which serves as a “reference point” for identifying, e.g., as described above, equivalent residues in other members of the family of which the reference enzyme is a member.
- the phrase “the amino acid corresponding to a reference residue in the mature human protein X” refers to residues equivalent (or homologous) to the reference residue of protein X in other members of the same protein family.
- the phrase refers to the reference residue itself.
- a “serine hydrolase” is a hydrolytic enzyme utilizing an active serine side chain to serve as a nucleophile in a hydrolytic reaction.
- This term includes native and synthetic serine hydrolases as well as enzymes engineered to perform the reverse reaction, e.g., for synthetic purposes.
- the family of serine peptidases is characterized by Bartlett and Rawlings (1994) Meth. Enzymol, 244: 19-61, Academic Press, S.D.
- the "alpha/beta serine hydrolases” are a family of serine hydrolyases based on structural homology to enzymes including wheat germ serine carboxypeptidase's II (see, e.g., Liam et al (1992) Biochemistry 31: 9796-9812; Ollis et al. (1992) Protein Engineering, 5: 197-211).
- aspartic proteases are proteases that are directly dependent on aspartic acid residues for catalytic activity.
- the family of aspartyl proteases is characterized in a number of publications known to those of skill in the art (see, e.g., Rawlings and Barrett, (1995) Meth. Enzymology, 248: 105-120, Academic Press, S.D.).
- cyste proteases is used herein consistently with conventional usage of those of skill in the art.
- the family of cysteine proteases is characterized in a number of publications known to those of skill in the art (see, e.g., Rawlings and Barrett, (1994) Meth. Enzymology, 224: 461-486, Academic Press, S.D.).
- metalloproteases is used herein consistently with the conventional usage of those of skill in the art.
- the family of metalloproteases is characterized in a number of publications known to those of skill in the art (see, e.g., Rawlings and Barrett, (1995)
- subtilisin type serine proteases refer to a family of serine hydrolyases based on structural homology to enzymes derived from Bacillus subtilus, including subtilisin BPN' (Bott et al. (1988) J. Biol. Chem. 263: 7895-7906; Siezen and Louise (1997) Protein Science 6: 501-523; Bartlett and Rawlings (1994) Meth. Enzymol, 244: 19-61, Academic Press, S.D.).
- Subtilisins are bacterial or fungal proteases which generally act to cleave peptide bonds of proteins or peptides.
- subtilisin means a naturally- occurring subtilisin or a recombinant subtilisin.
- a series of naturally-occurring subtilisins is known to be produced and often secreted by various microbial species. Amino acid sequences of the members of this series are not entirely homologous. However, the subtilisins in this series exhibit the same or similar type of proteolytic activity.
- This class of serine proteases shares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases.
- the subtilisins and chymotrypsin related serine proteases have a catalytic triad comprising aspartate, histidine and serine.
- subtilisin In the subtilisin related proteases the relative order of these amino acids, reading from the amino to carboxy terminus, is aspartate-histidine-serine. In the chymotrypsin related proteases, the relative order, however, is histidine-aspartate-serine. Thus, subtilisin herein refers to a serine protease having the catalytic triad of subtilisin related proteases.
- chymotrypsin serine protease family refers to a family of serine hydrolyases based on structural homology to enzymes including gamma chymotrypsin (Birktoft and Blow (1972) J. Molecular Biology 68: 187-240).
- a "dendritic polymer” is a polymer exhibiting regular dendritic branching, formed by the sequential or generational addition of branched layers to or from a core.
- dendritic polymer encompasses "dendrimers", which are characterized by a core, at least one interior branched layer, and a surface branched layer (see, e.g., Petar et al. Pages 641-645 In Chem.
- dendrimer is a species of dendrimer having branches emanating from a focal point which is or can be joined to a core, either directly or through a linking moiety to form a dendrimer. Many dendrimers comprise two or more dendrons joined to a common core. However, the term dendrimer is used broadly to encompass a single dendron.
- Dendritic polymers include, but are not limited to, symmetrical and unsymmetrical branching dendrimers, cascade molecules, arborols, dense star polymers, and the like.
- the PAMAM dense star dendrimers (disclosed in U.S. Patent 5,714,166) are symmetric, in that the branch arms are of equal length. The branching occurs at the nitrogen atom of a terminal amine group on a preceding generation branch.
- the lysine-based dendrimers are unsymmetric, in that the branch arms are of a different length.
- One branch occurs at the epsilon nitrogen of the lysine molecule, while another branch occurs at the alpha nitrogen, adjacent to the reactive carboxy group which attaches the branch to a previous generation branch.
- hyperbranched polymers e.g., hyperbranched polyols
- an “antibody” refers to a protein or glycoprotein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
- the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
- Light chains are classified as either kappa or lambda.
- Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
- a typical immunoglobulin (antibody) structural unit is known to comprise a tetramer.
- Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
- the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
- the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
- Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
- pepsin digests an antibody below (i.e. toward the Fc domain) the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to V H -CH1 by a disulfide bond.
- the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into an Fab' monomer.
- the Fab' monomer is essentially a Fab with part of the hinge region (see, Paul (1993) Fundamental Immunology, Raven Press, N.Y.
- antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically, by utilizing recombinant DNA methodology, or by "phage display” methods (see, e.g., Vaughan et al. (1996) Nature Biotechnology, 14(3): 309-314, and PCT/US96/10287).
- Preferred antibodies include single chain antibodies, e.g., single chain Fv (scFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.
- scFv single chain Fv
- carboxylic acids include mono-, oligo- and poly-saccharides as well as substances derived from monosaccharides by reduction of the carbonyl group (alditols), by oxidation of one or more terminal groups to carboxylic acids, or by replacement of one or more hydroxy group(s) by an hydrogen atom, an amino group, a thiol group or similar heteroatomic groups. It also includes derivatives of these compounds.
- saccharide is frequently applied to monosaccharides and lower oligosaccharides.
- Parent monosaccharides are polyhydroxy aldehydes H-[CHOH] n -CHO or polyhydroxy ketones H-[CHOH] justify-CO- [CHOH] m -H with three or more carbon atoms.
- the generic term "monosaccharide” denotes a single unit, without glycosidic connections to other such units. It also includes aldoses, dialdoses, aldoketoses, ketoses and diketoses, as well as deoxy sugars and amino sugars, and their derivatives, provided that the parent compound has a (potential) carbonyl group (see, e.g., McNaught (1996) Pure Appl. Chem.
- Carbohydrates also include, but are not limited to, oligosaccharides and polysaccharides (e.g. starch, cellulose, glycogen) and carbohydrate analogues (e.g., those in which OH have been replaced by H, F, NH 2 or NHC(O)CH 3 ).
- the term "soil” or “stain” refers to the accumulation of foreign material on a substrate of interest (e.g. a textile). The "soil” or “stain” may have no biological activity, but may serve to discolor, and/or degrade the underlying substrate.
- Typical stains or soils include, but are not limited to grass stains, blood stains, milk stains, egg, egg white, and the like.
- small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
- Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about
- near refers to a residue covalently attached to the "reference residue", either preceding or following that residue, or in van der Waals contact with the reference residue.
- Figure 1 illustrates a variety of chimeric molecules of this invention utilizing dendrimers as targeting moieties.
- Figure 2 illustrates SBL targeting an enzyme with an inhibitor.
- Figure 3 illustrates scheme 11 for synthesis of MTS-pyrazole 4.
- Figure 4 illustrates results of HLADH targeting assay for SBL-pyrazole chimeric molecules.
- Figure 5 A, Figure 5B, Figure 5C, and Figure 5D illustrate results of HLADH degradation assay for SBL-pyrazole chimeric molecules.
- Figure 6 shows HLADH activity for HLADH/ AP mixtures with and without S166C- pyrazole.
- Figure 7 shows AP activity for HLADH/ AP mixtures with and without S166C-pyrazole.
- Figure 8 shows HLADH activity for HLADH/AP mixtures with and without S166C-pyrazole.
- Figure 9 shows AP activity for HLADH/AP mixtures with and without S166C-pyrazole.
- Figure 10 shows HLADH degradation by substoichiometric pyrazole-CMMs.
- Figure 11 shows HLADH degradation by pyrazole-CMMs in the presence of alkaline phosphatase
- Figure 12 illustrates alkaline phosphatase degradation by pyrazole-CMMs in the presence of HLADH.
- Figure 13 shows 11 mono- and disaccharide methanethiosulfonates that were prepared.
- Figure 14A, Figure 14B, and Figure 14C illustrate selective lectin degradation by sugar-modified GG36-WT.
- Figure 15 A, Figure 15B, Figure 15B, and Figure 15D illustrate time course plots of the formation of ⁇ 3000 MW protein fragments during a lectin assay.
- Figure 16 illustrates synthesis scheme 7 for the synthesis of biotin-MTS reagent 1.
- Figure 17 illustrates a standard enzyme linked immunosorbent assay (ELISA)-technique for assaying targeting of biotinylated CMMs to anti-biotin.
- ELISA enzyme linked immunosorbent assay
- Figure 18 illustrates a targeting assay for anti-biotin using using hapten modified subtilisins in a 96-well plate.
- Figure 19 plot of anti-biotin degradation by biotin-CMM as a function of time.
- the chimeric molecules are catalytic antagonists of a target molecule.
- the catalytic antagonists of this invention preferably comprise a targeting moiety attached to an enzyme that degrades the molecule specifically bound by the targeting moiety.
- the catalytic antagonists of this invention thus bind to a target recognized by the targeting moiety (e.g. a receptor) the enzyme component of the chimera then degrades all or part of the target. This preferably resulting in a reduction or loss of activity of the target and release of the chimeric molecule.
- the chimeric molecule is then free to attack and degrade another target molecule.
- a chimeric molecule of this invention is free to attack and degrade essentially a limitless number of targets.
- the antagonists of this invention are thus catalytic in nature being effectively regenerated (rendered available again) after degrading each substrate molecule (target).
- the activity of the catalytic antagonists of this invention is thus essentially sub-stoichiometric.
- the catalytic antagonists of this invention are effective in far lower concentrations than chimeric molecules or traditional inhibitors. Consequently formulations (e.g. detergents) comprising the catalytic inhibitors of this invention can utilize significantly lower concentrations of inhibitor and can be fabricated at lower cost. In in vivo applications, the catalytic inhibitors of this invention because they offer greater activity at lower concentration, are expected to show longer effective serum half-life and lower toxicities than "traditional" chimeric molecules.
- the catalytic antagonists of this invention are useful in a wide variety of contexts where it is desired to degrade a target molecule and/or inhibit the activity of that target molecule.
- the catalytic antagonists can be used to specifically target and degrade a particular molecule.
- the chimeric molecules of this invention can be utilized to specifically target and degrade a component of a soil (e.g. a protein component, a lipid component, etc.).
- biochemical synthetic processes e.g. in analytic or industrial preparations, in bioreactors, etc. to specifically degrade particular preselected molecules.
- the catalytic antagonist of this invention comprises, as a targeting moiety, a substrate for the enzyme mediating the activity (e.g. a glycosyltransferase).
- the enzyme (receptor) in the reactor binds the targeting moiety and the enzymatic component of the chimera (e.g. a hydrolase) degrades the enzyme reducing or eliminating its activity and also freeing itself from the enzyme binding site whereby it is free to attack another target enzyme.
- chimeric molecules of this invention having, e.g. targeting moieties directed against lectins present on bacterial surfaces attached to, e.g. Upases, or hydrolases, are effective antimicrobial agents and can be used in a wide variety of disinfectants.
- the chimeric molecules of this invention can be used to bind and antagonize/inhibit a wide variety of receptors and/or enzymes, and/or intermediary signaling molecules.
- a wide variety of drugs act by inhibiting the activity of cellular receptors.
- antiestrogens e.g. tamoxifen
- beta blockers e.g.
- the catalytic antagonists of this invention effectively degrade the receptor.
- the receptor is unlikely to function again, absent some repair mechanism.
- the catalytic antagonists of this invention will produce a far greater degree of activity and/or duration of activity than "traditional" competitive inhibitors.
- an enzyme e.g. an intracellular enzyme
- catalytic antagonists of this invention can be used to degrade target enzymes as well. In this instance, it is preferably to use, as the targeting moiety, a molecule that is not degraded or altered by the target enzyme.
- Known competitive inhibitors of enzymes make good targeting moieties in this context.
- this invention include chemical antagonists (e.g. of receptors and/or enzymes) comprising a targeting moiety that binds to the receptor or enzyme attached to an enzyme that degrades the cognate ligand that binds to that enzyme and/or receptor.
- the inhibitor of the enzyme or receptor binds and anchors the enzyme comprising the chimeric molecule to the target enzyme or receptor.
- the enzyme degrades is and thereby blocks its activity on the receptor.
- the process is "catalytic" with no permanent change to the chimeric molecule.
- the catalytic antagonists of this invention are chemically coupled chimeric molecules.
- the targeting moiety preferably coupled, directly or through a linker, to either terminus of the enzyme (the amino or carboxyl terminus or through an R group of the terminal amino acid), or more preferably, is coupled, directly or through a linker, to a non-terminal amino acid in the enzyme.
- the catalytic antagonists of this invention comprise a chemically modified mutant (CMM) enzyme.
- CCM chemically modified mutant
- a chemically modified mutant enzyme is an enzyme in which a native amino acid residue is replaced with a different amino acid residue (e.g. cysteine, affording a reactive site suitable for coupling the targeting moiety.
- preferred chimeric molecules of this invention are chemically coupled molecules rather than fusion proteins.
- the use of chemically coupled targeting moieties in this invention affords a number of advantages.
- the targeting moiety is not limited to a peptide or protein, but rather can be any of a number of ligands including, but not limited to, known drugs, vitamins, carbohydrates, lectins, and the like. Because the targeting moieties are typically smaller than proteins, they are less immunogenic and show greater tissue penetration. In addition, because the targeting moieties are often various small organic molecules, they retain their conformation and specificity in a physiological context and are typically less subject to degradation in vivo.
- the chimeric molecules of this invention offer a number of other advantages. Because they are chemically conjugated using a "standard" chemistry, they are easier to make and/or to vary.
- the molecules are smaller than typical "therapeutic" fusion proteins (e.g. immunotoxins) and are expected to have increased serum half-life.
- the molecules actually destroy/degrade existing receptors and/or enzymes, a single dosage is expected to have a longer-lasting effect since the subject organisms must actually replace the receptor and/or enzyme to restore that functionality.
- the catalytic antagonists of this invention can be regarded as enzymes that have been "redirected'-' so that they either act on a non-native substrate (for the enzymatic component) or, more typically, so that the enzymatic activity is localized at the site of the target molecule.
- this invention provides an enzyme having altered substrate specificity where the enzyme is a component of a chimeric molecule comprising a targeting moiety attached to a subsite comprising the substrate binding site of the enzyme.
- targeted chimeric molecules are designed to position the targeting moiety/domain some distance away from active sites of interest in the effector moiety. It was generally believed that a targeting moiety located too close to an active site of the effector moiety would interfere with proper functioning of the effector (e.g. via steric hindrance).
- targeting moieties comprising the chimeric molecules of this invention can be coupled to amino acid residues comprising a substrate binding site of the enzyme. Moreover, attachment of the targeting moiety to an amino acid residue in the substrate binding site of the enzyme results in the substrate binding site being closely juxtaposed to the target bound by the targeting moiety.
- Using chemically conjugated mutants according to the methods of this invention provides a versatile method of directing a single enzyme to any target simply by changing the chemical moiety. This is a substantial advantage over traditional methods where extensive modification (e.g. by mutagenesis techniques) was required to make a particular target-specific enzyme.
- the activity of the enzyme is thus “redirected” in one or both of two ways: First the activity of the enzyme can be “spatially localized” by binding of the targeting moiety to a particular preselected target. Thus, the enzyme may be specifically directed to a particular cell type, a particular enzyme, a particular receptor, etc. Second, by virtue of alterations in the enzyme produced by the presence o ⁇ the targeting molecule and/or by virtue of the fact that the targeting molecule brings the substrate binding site in close proximity to the target, the enzyme can show significant activity against a target that is not its usual substrate.
- the targeting moiety can be selected to redirect/localize the enzyme to a particular target for selective degradation.
- the targeting moiety can be selected to specifically bind to a particular class of "soil” (e.g. egg) and thereby direct and appropriate degradative enzyme (e.g. a protease) to that substrate.
- the retargeted enzyme can comprise a targeting moiety that directs the enzyme to a particular target cell (e.g. a tumor cell) where the retargeted enzyme (e.g. a thymidine kinase (tk)) activates a particular drug (e.g. a cytotoxin such as ganclovir).
- a particular target cell e.g. a tumor cell
- the retargeted enzyme e.g. a thymidine kinase (tk)
- a particular drug e.g. a cytotoxin such as ganclovir
- the enzyme may be retargeted to a cell that contains an overabundance of a particular metabolite (e.g. as in storage diseases such as Tay Sachs disease).
- the redirected enzyme affords the "missing" enzymatic activity thereby treating the condition.
- catalytic antagonists can be retargeted enzymes, but are not necessarily so. Conversely, retargeted enzymes may act as catalytic antagonists, but there are retargeted enzymes that are not necessarily catalytic antagonists.
- the retargeted enzymes and catalytic antagonists are created by selecting a targeting moiety, selecting an enzyme (an effector moiety) and chemically conjugating the two to form a chimeric molecule.
- any cognate binding partner of a target e.g. a receptor and/or an enzyme and/or a lectin
- a targeting moiety in the molecules of this invention.
- molecules that are not cognate binding partners, but that are specifically bound by the target molecules e.g. receptors or an enzymes
- targeting moieties in the chimeric molecules of this invention.
- targeting moiety depends on the application for which the chimeric molecule (e.g. catalytic antagonist) is to be utilized.
- Targeting moieties can be grouped and/or identified according to a wide variety of classification schemes. Thus, for example they can be grouped according to type of molecule, e.g.
- Prefe ⁇ ed targets include, but are not limited to receptors, enzymes, and lectins. In some instances it is simple to refer to the targeting moiety that binds to one of these targets.
- serotonin or a serotonin analogue may be a targeting moiety.
- targeting moieties can be refe ⁇ ed to/identified by the target to which they bind.
- a serotonin analogue as targeting moiety is encompassed by a drug or compound that specifically binds to a serotonin receptor.
- Receptors provide highly effective targets, particularly for the catalytic antagonists of this invention.
- Receptors typically specifically bind a cognate ligand and are involved in a wide variety of biological processes.
- receptors mediate signaling or the influx or efflux of molecules from a cell.
- transducers of signals receptors are involved in a wide variety of processes including, but not limited to regulation of growth and morphology/differentiation, gene expression and production of particular molecules, cell proliferation, elements of the immune response, various biological cascades (e.g. the inflammatory response, the clotting response, etc.) and the like.
- receptors have long been recognized as good targets for drugs and a wide variety of drugs are agonists and/or antagonists of particular receptor activity (see, e.g., Table 1).
- these drugs are relatively small organic molecule and, as such, are good candidates as targeting moieties for the chimeric molecules of this invention.
- the targeting moiety need not be a known pharmaceutical.
- the first class includes receptors that penetrate the plasma membrane and have intrinsic enzymatic activity.
- Such receptors include, but are not limited to, those that are tyrosine kinases (e.g. PDGF, insulin, EGF and FGF receptors), tyrosine phosphatases (e.g. CD45 [cluster determinant-45] protein of T cells and macrophages), guanylate cyclases (e.g. natriuretic peptide receptors), , and serine/threonine kinases (e.g.
- tyrosine kinases e.g. PDGF, insulin, EGF and FGF receptors
- tyrosine phosphatases e.g. CD45 [cluster determinant-45] protein of T cells and macrophages
- guanylate cyclases e.g. natriuretic peptide receptors
- serine/threonine kinases e.g.
- PKA cAMP-dependent protein kinase
- PKC protein kinase C
- MAP kinases activin and TGF- ⁇ receptors
- TGF- ⁇ receptors cAMP-dependent protein kinase
- PKA cAMP-dependent protein kinase
- PLC protein kinase C
- MAP kinases activin and TGF- ⁇ receptors
- the proteins encoding receptor tyrosine kinases typically contain four major domains: an extracellular ligand binding domain, an intracellular tyrosine kinase domain, an intracellular regulatory domain, and a transmembrane domain.
- the amino acid sequences of the tyrosine kinase domains of RTKs are highly conserved with those of cAMP-dependent protein kinase (PKA) within the ATP binding and substrate binding regions.
- PKA cAMP-dependent protein kinase
- Some RTKs have an insertion of non-kinase domain amino acids into the kinase domain termed the kinase insert.
- RTK proteins are classified into families based upon structural features in their extracellular portions (as well as the presence or absence of a kinase insert) which include the cysteine rich domains, immunoglobulin-like domains, leucine-rich domains, Kringle domains, cadherin domains, fibronectin type III repeats, discoidin I-like domains, acidic domains, and EGF-like domains. Based upon the presence of these various extracellular domains the RTKs have been sub-divided into at least 14 different families.
- RTKs include, but are not limited to I EGF receptor, NEUHER2, HER3, insulin receptor, IGF-1 receptor, PDGF receptors, c-Kit, FGF receptors, vascular endothelial cell growth factor (VEGF) receptor, hepatocyte growth factor (HGF) and scatter factor (SC) receptors, the neurotrophin receptor family (trkA, trkB, trkC) and NGF receptor, and the like.
- the second class includes receptors that are coupled, inside the cell, to GTP- binding and hydrolyzing proteins (termed G-proteins).
- G-protein coupled receptors are a superfamily of integral membrane proteins that are typically characterized by seven hydrophobic domains which are of sufficient length (typically 20-28 amino acid residues) to span the plasma membrane. Examples of this class include, but are not limited to the -adrenergic receptors, odorant receptors and receptors for peptide hormones (e.g. glucagon, angiotensin, vasopressin and bradykinin).
- the third class includes receptors that are found intracellularly and that, upon ligand binding, migrate to the nucleus where the ligand-receptor complex directly affects gene transcription.
- receptors include, but are not limited to steroid/thyroid hormone receptor superfamily (e.g. glucocorticoid, vitamin D, retinoic acid and thyroid hormone receptors). This is a class of proteins that reside in the cytoplasm and bind the lipophilic steroid/thyroid hormones.
- hormone-receptor complex Upon binding ligand the hormone-receptor complex translocates to the nucleus and binds to specific DNA sequences termed hormone response elements (HREs). The binding of the complex to an HRE results in altered transcription rates of the associated gene.
- HREs hormone response elements
- a 2 receptor agonists see, e.g., U.S. Patent 6,026,317)
- 5HT1 receptor agonists or antagonists see, e.g., U.S. Patent 6,025,374 and 6,025,367
- N- methyl-D-aspartate (NMD A) receptor blockers for the prevention of atherosclerosis see, e.g., U.S. Patent 6,025,369
- modulators of peroxisome proliferator activated receptor- gamma see, e.g., U.S. Patent 6,022,897)
- endothelin receptor antagonists see, e.g., U.S.
- Patents 6,022,886, 6,020,348) human growth hormone variants having enhanced affinity for human growth hormone receptor at site 1 (see, e.g., U.S. Patent 6,022,711), antagonists of the human neuronal nicotinic acetylcholine receptor (see, e.g, U.S. Patent 6,020,335), platelet GPIIb/IIIa receptor antagonists (see, e.g., U.S. Patent 6,022,523), adenosine receptor agonists (see, e.g., U.S. Patent 6,020,321), interleukin receptor (e.g.
- IL-2R, IL-4R, IL-6R, IL-8R, IL-10R, IL-13R, etc. antagonists, binding agents specific for growth factor receptors (e.g. EGF, TGF and analogues or mimetics thereof), binding agents specific for IgA receptor (see, e.g., U.S. Patent 6,018,031), agonists of the strychnine insensitive glycine modulatory site of the N-methyl-D-aspartate receptor complex (see, e.g., U.S. Patent 6,017,957), integrin receptor antagonists (see, e.g., U.S.
- Patent 6,017,926) androgen receptor modulator compounds (see, e.g., U.S. Patent 6,017,924), PCP receptor ligands (see, e.g., U.S. Patent 6,017,910), azole peptidomimetics as thrombin receptor antagonists (see, e.g., U.S. Patent 6,017,890), NPY Y2-receptor agonists (see, e.g., U.S. Patent 6,017,879), receptor activators of NF- ⁇ B (see, e.g., U.S.
- Patent 6,017,729) antagonists of the TNF receptor, somatostatin receptor-binding agents (see, e.g., U.S. Patent 6,017,509), human histamine H 2 receptor, bradykinin binding agents (see, e.g., U.S. Patent 6,015,812 ), glutamate receptor antagonist (see, e.g., U.S. Patent 6,015,800), imidazoline receptors, transferrin receptors, benzodiazepine receptor binding agents (see, e.g., U.S. Patent 6,015,544), gaba brain receptor ligands (see, e.g., U.S. Patent 6,013,799), neurotensin NT1 and NT2 receptors, CXCR2 receptors, CCR5 receptors, macrophage mannose receptors, and the like.
- TNF receptor see, e.g., U.S. Patent 6,017,509
- human histamine H 2 receptor see, e.g
- the targeting moieties used in the chimeric molecules of this invention are moieties specifically bound by enzymes or antibodies.
- enzymes or antibodies A wide variety of enzymes, their substrates and competitive inhibitors thereof are known to those of skill in the art. Moreover, many of these enzymes provide good targets for drug in a wide variety of pathologies.
- caspases are a remarkable and intricately regulated network of enzymes that can trigger cell suicide in animals from yeast and worms to humans.
- Caspases are known to mediate programmed cell death in a number of diseases, including ischemic brain injury, or stroke. It is believed that the cardiac cell death that occurs during heart
- YVAD-cmk an experimental caspase inhibitor known as YVAD-cmk blocks this biochemical cascade and also protects heart tissue, dramatically reducing the amount of myocardial deaths by over 30 percent.
- Catalytic antagonists of this invention comprising caspase-specif ⁇ c agents as targeting moieties attached to a protease (enzyme) can specifically target and degrade the caspase. It is expected this will offer protection of heart tissue during and after myocardial infarction and brain tissue during and after stroke.
- Agents that specifically bind to caspases e.g. YVAD-cmk, and various protected caspase substrates are known to those of skill in the art.
- the enzyme GARFT (Glycinamide Ribonucleotide Formyl Transferase) is an enzyme in a biochemical pathway through which tumor cells synthesize purines, essential components of DNA. Blocking the action of GARFT inhibits purine synthesis and subsequent tumor DNA molecule construction. With the exception of liver cells, all normal human tissues can obtain purines via an alternative pathway (purine salvage pathway). Inhibitors of GARFT will show selectivity for tumor cells and less significant bone ma ⁇ ow toxicity than other chemotherapeutic agents.
- a catalytic antagonist of this invention comprising a GARFT targeting moiety attached to a protease capable of degrading GARFT is expected to show similar tumor selectivity.
- AG2037 produced by Agouron
- AG2037 has been engineered using structure-based design to exhibit potent and selective inhibition of GARFT but to avoid binding to mFBP, a membrane protein believed to be important in the side- effects of earlier GARFT inhibitors.
- AG2037 is well tolerated in a variety of mouse cancer models and demonstrates broad-spectrum antitumor efficacy, at least equal to that of paclitaxel when studied in the same tumors grown in mice.
- DHODH dihydroorotate dehydrogenase
- UMP uridylate
- Hoechst Leflunomide
- FDA Food and Drug Administration
- DHODH-specific agents can be used as a targeting moiety attached to an enzyme that degrades the DHODH enzyme and provides a similar therapeutic result.
- Another known inhibitor, Brequinar sodium has shown efficacy in many animal models of immunosuppression, but was not successful in clinical trials for transplantation, apparently due to a na ⁇ ow therapeutic window.
- a targeting moiety in a catalytic antagonist of this invention it is expected that the therapeutic window will be improved because the molecule will be effective in lower dosages.
- conversion of drugs that act as competitive inhibitors into catalytic antagonists in accordance with this invention will show an improved therapeutic window due to their higher efficacy at lower concentration.
- the catalytic antagonists of this invention are useful in the treatment of hereditary emphysema.
- the inherited form of emphysema is called alpha- 1 proteinase inhibitor deficiency or "alpha - one" for short.
- People with this disease have a deficiency in a major protein, alpha- 1 proteinase inhibitor.
- Alpha- 1 proteinase inhibitor is a major protein in the blood and is produced primarily in the liver cells but also by some white blood cells. It protects the lung by blocking the effects of powerful enzymes called elastases.
- Elastase is normally carried in white blood cells and protects the delicate tissue of the lung by killing bacteria and neutralizing tiny particles inhaled into the lung. Once the protective work of this enzyme is finished, further action is blocked by the alpha- 1 proteinase inhibitor. Without alpha- 1 proteinase inhibitor, elastase can destroy the air sacs of the lung.
- catalytic antagonists of this invention comprising an alpha- 1 proteinase binding moiety attached (e.g. the drug called Prolastin) to, e.g. a protease, will degrade alpha- 1 proteinase affording similar or better therapeutic benefit.
- an alpha- 1 proteinase binding moiety attached e.g. the drug called Prolastin
- Antibodies also provide useful targets for the catalytic antagonists of this invention.
- a catalytic antagonist that targets and antagonizes (e.g. degrades) ⁇ -Gal epitope specific antibodies is expected to significantly reduce an immune response (e.g. to a xenotransplant).
- the ⁇ -Gal epitope can be used as a targeting moiety in a chimera of this invention. It may be attached to a protease (e.g. a subtilisin, a pepsin, etc.) and when it is bound by the antibody it will degrade that antibody thereby inhibiting the antibody-mediated immune response.
- the catalytic antagonist can use as a targeting moiety the MHC (or component thereof) of the xenotransplant.
- the enzymatic component is a hydrolase (e.g. a protease)
- the catalytic inhibitor will specifically digest the receptor on effector cells of the immune system (e.g. cytotoxic T lymphocytes (CTLs) only on those cells specifically directed against the xenotransplant.
- CTLs cytotoxic T lymphocytes
- antibodies that are good targets for the catalytic inhibitors of this invention are antibodies produced in auto-immune responses and/or other allergic responses.
- the targeting moiety is a molecule bearing an epitope recognized by the antibodies mediating the autoimmune or allergic response. Degradation of the antibody by, e.g. a hydrolase attached to the targeting moiety will reduce the pathologic symptoms associated with the autoimmune response.
- Specific allergens and substrates recognized by antibodies in various autoimmune responses are well known to those of skill in the art and can readily incorporated into the chimeric molecules of this invention.
- targeting moieties are selected that bind to particular lectins.
- Lectins are proteins obtained from many plant, animal, and bacterial sources that have binding sites for specific mono or oligosaccharides. Lectins such as concanavalin A and wheat germ agglutinin are widely used as analytical and preparative agents in the study of glycoproteins.
- Lectins are also present on the surfaces of eukaryotic and bacterial cells. In eukaryotic cells, lectins are often involved in cell-cell interactions. In bacterial cells, lectins often mediate adhesion of the bacterium to its target/host and, in many cases, such adhesion is required for the bacterium to infect the host cell.
- Biofilm formation is also problematic in a wide variety of commercial synthetic systems. Often fermentation vessels and other bioreactors are contaminated by biofilms. Biofilms also growing in and contaminate apparatus used for many chemical processes particularly those involving "digestible" organic reagents. Thus, biofilms often contaminate filters, conduits, separators and other devices.
- the catalytic antagonists of this invention can be used to inhibit/degrade various lectins.
- a catalytic antagonist comprising a sugar or oligosaccharide attached to a hydrolase will specifically target and degrade a lectin that binds the target molecule.
- Such a catalytic antagonist is illustrated in the examples.
- Degradation of lectins/adhesins on a bacterial surface will interfere with the bacteria's ability to bind to a surface and thereby prevent the bacterium from entering a host cell or from forming a biofilm.
- ganglioside an acid glycosphingolipid and invade host cells.
- cholera toxin an enterotoxin produced by Vibrio cholerae, which is known to bind ganglioside GM1.
- Other ganglioside-binding bacterial toxins include Tetanus toxin (GDlb), botulinum toxins (GTlb and GQlb) and delta toxin produced by Clostndium perfringens (GM2). Shiga toxin produced by Shigella dysenteriae and Vero toxin produced by enterohaemo ⁇ hagic E.
- coli bind to neutral glycosphingolipids having an alpha- 1,4 galabiose moiety in the sugar chain, such as galabioside (Ga2Cer) and ceramide trihexoside (Gb3Cer). Many pathogenic bacteria also bind to glycosphingolipids of host cell surface for colonization and infection. Thus, uropathogenic E. coli which cause urinary tract infections can bind to glycosphingolipids having an alpha- 1,4 galabiose moiety at the non- reducing end of the sugar chain (Gb3Cer, etc). Uropathogenic ⁇ .
- E. coli can also bind to globoside (Gb4Cer) and Forssman glycolipid, both of which have an alpha- 1,4 galabiose moiety internally in a sugar chain.
- Gb4Cer globoside
- Forssman glycolipid both of which have an alpha- 1,4 galabiose moiety internally in a sugar chain.
- E. coli binds to glycosphingolipids by pili that exist on the bacterial cell surface and are similar to fibers or hairs. On the top of pili, there is an adhesin characterized as a lectin.
- adhesin characterized as a lectin.
- types of adhesin with respect to their sugar specificity, have been identified. They are the type I adhesin of E. coli that are mannose specific, type P adhesin, also of E. coli, specific for alpha- 1,4 galabiose moiety, and type S adhesin of E.
- Propionibacterium which causes skin disease, recognizes the lactosyl moiety of glycosphingolipids as a binding epitope. These bacteria can bind strongly to lactosylceramide and also bind to isoreceptors such as asialo GM1 (GA1) and asialo GM2 (GA2). Because almost all glycosphingolipids contain a common lactosyl moiety, Propionibacterium may be assumed to bind almost all glycosphingolipids.
- the bacteria cannot bind to any glycosphingolipids composed of a dihydroxy base and nonhydroxy fatty acid in ceramide, even though these contain a lactosyl moiety. This fact indicates that the binding epitope of the bacteria also depends on the ceramide structure in addition to the lactosyl moiety in sugar chain. Neisseria gonorrhoeae, which cause gono ⁇ hoea, also bind glycosphingolipids having a lactosyl moiety.
- catalytic antagonists having targeting moieties that specifically bind various glycosphingolipids and/or various adhesins (e.g. mannose specific type I adhesin of E. coli, alpha- 1,4 galabiose specific type P adhesin of E. coli, and sialylgalactose type S adhesin of E. coli) attached to enzymes that degrade the glycosphingolipids (e.g. glycosidases, cerebrosidases, etc.) will act to prevent bacterial infections and are expected to provide effective therapeutics to block acute effects of bacterial-produced toxins (e.g. cholera toxin).
- various adhesins e.g. mannose specific type I adhesin of E. coli, alpha- 1,4 galabiose specific type P adhesin of E. coli, and sialylgalactose type S adhesin of E. coli
- Lectins are also implicated in various inflammatory processes (e.g. inflammatory processes associated with rheumatoid arthritis, arthritis, septic shock, myocardial infarction., etc.).
- Lec-CAMs or selectins are expressed on the surfaces of endothelium, leukocytes and platelets and influence leukocyte- endothelial adhesion at sites of inflammation.
- GMP-140 P-selectin
- ⁇ LAM-1 E-selectin
- LAM-1 L-selectin regulates lymphocyte binding to high endothelial lymph node venules, the surface of neutrophils and lymphocytes to localize these cells to injury.
- the integrins a family of adhesion molecules composed of heterodimers of ⁇ and ⁇ subunits; act in regulation of cell-matrix and cell-cell adhesion. These molecules are transmembrane in structure, thus linking or "integrating" exterior/surface stimuli to the internal cell cytoskeleton.
- R2 integrins also known as CDl 1/CD18 molecules confer adhesion specificity, mediate activation of phagocytic cells by chemotactic stimuli.
- the surface expression of integrins e.g. MO-1, leukocyte function antigen- 1 (LFA-1) and gpl50,95; assist in localization of phagocytes to injury sites; deficiency states result in enhanced susceptibility to bacterial infection.
- the intercellular adhesion molecule- 1 (ICAM-1): assists in localization of leukocytes to tissue injury; expressed on surface of cytokine stimulated endothelium and leukocytes; binds to LFA-1 and MO-1 present on cell membranes of neutrophils and macrophages.
- the vascular cell adhesion molecule- 1 (VCAM-1) binds VLA-4 leukocyte receptor on lymphocytes, monocytes, eosinophils, basophils.
- the targeting moiety can be a lectin that will specifically direct the catalytic antagonist (or redirected enzyme) to a sugar or sugar-bearing target.
- the molecules can be directed to the sugars present on and characteristic of particular bacteria.
- the sugar-targeted catalytic antagonist will make an effective microbicide.
- such molecules can be targeted by using simple sugars, or oligosaccharides and the like as targeting moieties in the chimeric molecules of this invention.
- dendrimers can also be used as targeting moieties.
- multiple functionalization of the enzyme e.g. either a catalytic antagonist or a redirected enzyme
- dendrimeric targeting moieties whereby multiple branched linking structures can be employed to create a polyfunctionalized enzyme (chimeric molecule).
- multiple glycosylation including multiple mannose-containing chimeras and varied sugar moieties can be created.
- This could confer the benefit of increased affinity for, and increased binding affinity between, lectins and the targeted enzyme (e.g. a hydrolase).
- the targeted enzyme e.g. a hydrolase.
- This would also permit multiple concurrent targeting of sites, for instance by incorporating multiple biotin molecules into a targeting moiety that would elicit multiple concu ⁇ ent biotin-avidin interactions.
- the dendrimer targeting moieties (before coupling to the enzyme) would preferably include methanethiosulfonates with simple branching such as:
- the redirected enzymes describe herein can be used in a variety of drug delivery strategies.
- the targeting moiety can be directed to specifically bind to a particular cell type or tissue (e.g. a tumor cell).
- the enzymatic component can be selected for an activity that converts a (e.g. non-toxic prodrug) to an active form (e.g. a cytotoxin).
- the retargeted enzyme of this invention thus localizes the activity of the prodrug/drug to the cells bound by the chimeric molecule.
- Numerous cell-specific markers are known to those of skill in the art.
- thymidine kinase e.g., Herpes simplex thymidine kinase (HSVTK) or Varicella zoster thymidine kinase (VZVTK)
- thymidine kinase assists in metabolizing antiviral nucleoside analogues to their active form are therefore useful in activating nucleoside analogue precursors (e.g., AZT or ddC) into their active form.
- the redirected enzyme of this invention can be a thymidine kinase targeted, for example to a cell expressing a CCR5 and/or a CCR3 receptor (and hence likely to be susceptible to HIV infection).
- the tk re-directed to these cells can activate AZT or ddC precursors into their active form.
- the tk enzyme can be directed to a tumor cell (e.g. via a tumor specific antigen). Treatment with ganclovir then results in death of the tumor cell.
- Other examples of prodrugs that can be converted to their active form using the redirected enzymes of this invention include, but are not limited to prodrugs of 5-FU or inhibitors of dihydropyrimidine dehydrogenase (DPD) (GW 776C85).
- prodrug phosphenytoin a relatively soluble prodrug that is converted by phosphatase to relatively insoluble phenytoin an active anticonvulsant.
- depivefrin is converted by esterase to epinephrine, an adrenergic, useful in the treatment of glaucoma.
- the re-directed enzymes of this invention can also act as "self-protected" polypeptides particularly when utilized as in vivo therapeutics.
- the organic molecule (e.g. the targeting moiety) component of the chimeric molecule can sterically shield and protect the effector (enzyme) component of the chimeric molecule.
- the idea of this is that bulky groups attached near to key positions on the chimeric molecule would hinder the attack of another reagent on, e.g. cleavage sites in the remainder of the chimera and therefore prolong its lifetime.
- sugars on proteins increase their stability to proteinases, i.e., the proteinase can't get in to cleave its prefe ⁇ ed amide bond because a sugar is blocking it (see, e.g., Rudd et al. (1994) Biochemistry, 33: 17-22).
- the organic molecule can perform "double duty" providing both a targeting functionality as well as a protective function.
- the re-directed enzymes of this invention can be utilized in enzyme replacement therapy, particular in the treatment of storage diseases.
- Storage diseases are caused by the increased accumulation of metabolic products (e.g., lipids, proteins, and complex carbohydrates) due to either the inactivity of an enzyme that degrades the products or the hyperactivity of an enzyme that creates the products.
- Storage disease include but are not limited to glycogen storage disease I, GM1 gangliosidoses, MPS IV B (Morquio B), GM2 gangliosidoses (O, B, AB, Bl variants), Niemann-Pick disease (A, B, and C), Metachromatic leukodystrophy (arylsulfatase A and SAP-1 deficient), Krabbe disease, Fabry disease, Gaucher disease, Farber disease, Wolman disease (cholesterol ester storage disease), MPS I (Hurler and Scheie syndromes), MPS II (Hunter syndrome), MPS III A, C, and D (Sanfilippo A, C, and D), PS III B (Sanfilippo B), MPS IV A (Morquio A), MPS VI (Maroteaux-Lamy syndrome), MPS VII (beta-glucuronidase deficiency), Multiple sulfatase deficiency, Mucolipidosis I (Sialidosis), Muco
- Gaucher's disease can be treated by use of a glucocerebrosidase targeted to spleen cells.
- superoxide dismutase can be targeted to the liver as an anti-oxidant, and so forth.
- any enzyme can be utilized in the chimeric molecules of this invention.
- enzymes are selected that are capable of degrading the substrate specifically bound by the targeting moiety.
- Such enzymes include, but are not limited to proteases, cellulases, nucleases (exo- and endo-), amylases, lipases, aldolases, ketolases, glycosidases, and the like.
- the chimeric molecule is an enzyme whose activity is directed to a new location and/or substrate, the enzyme can be , but need not necessarily be an enzyme that degrades its substrate.
- the redirected enzymes can also include enzymes such as isomerases, oxidases, oxidoreductases, ligases, transferases, and the like.
- Prefe ⁇ ed enzymes for use in the catalytic antagonists of this invention are the hydrolases.
- Particularly prefe ⁇ ed enzymes for use in the catalytic antagonists of this invention are the serine hydrolases.
- the serine hydrolases are a class of hydrolytic enzymes characterized by a hydrolytic enzymes that posses a catalytic triad composed of a serine, histidine and a carboxylate amino acid (either aspartic or glutamic acid), and which catalyze the hydrolysis, and microscopic reverse reactions thereof, of carboxylic acid derivatives including, but not restricted to, esters, peptides and amides.
- Prefe ⁇ ed serine hydrolases comprising this invention include the trypsin- chymotrypsin proteases, the subtilisin proteases, and the alpha/beta hydrolases.
- the enzyme is protease, more preferably a subtilisin (e.g. a Bacillus lentis subtilisin).
- subtilisin is a serine endoprotease (MW ⁇ 27,500) which is secreted in large amounts from a wide variety of Bacillus species.
- the protein sequence of subtilisin has been determined from at least four different species of Bacillus (see, e.g., Markland et al.
- subtilisin BPN' from B. amyloligoefaciens
- subtilisin containing covalently bound peptide inhibitors Robottus, et al. (1972) Biochemistry 11: 2439-2449
- product complexes Robottus et al. (1972) Biochemistry 11 : 4293-4303
- transition state analogs Mol. Cell.
- hydrolases for use in this invention include, but are not limited to ⁇ / ⁇ hydrolases, trypsin/chymotryspsin families of serine hydrolase enzymes, aspartyl proteases, cysteine proteases, metalloproteases, lysozymes and other glycosidases etc.
- the chimeric catalytic antagonists and/or redirected enzymes of this invention are made by chemically conjugating the desired enzyme (directly or through a linker) to the targeting moiety. While many strategies are known for preparing chemically conjugated chimeric molecules (see, e.g., European Patent Application No. 188,256; U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; 4,589,071; 4,545,985 and 4,894,443; Borlinghaus et al. (1987) Cancer Res. 47: 4071-4075; Thorpe et al.
- the targeting moiety is derivatized/functionalized with a reactive group that can react with an available R group (e.g. NH 2 , N, NH, OH, COOH, SH, etc.) on an amino acid residue comprising the enzyme.
- an available R group e.g. NH 2 , N, NH, OH, COOH, SH, etc.
- the targeting moiety is derivatized as a methanethiosulfonate reagent that can then react with the -SH in a cysteine to provide the targeting moiety coupled in place of the thiol hydrogen on the cysteine.
- the coupling can be direct or through a linker.
- the cysteine to which the targeting moiety is attached is a native cysteine in the enzyme, however, in prefe ⁇ ed embodiments the cysteine is a cysteine substituted for a different native amino acid residue in the enzyme.
- the enzyme so modified is, optionally, refe ⁇ ed to as a mutant enzyme.
- Chimeric molecules of this invention in which the targeting molecule is chemically coupled to a mutant enzyme are, optionally, refe ⁇ ed to as Chemically Modified Mutants (CMMs).
- the location (residue) in the enzyme for attachment of the targeting moiety is identified. Where this residue is not already a cysteine, a cysteine is substituted for the native residue.
- the targeting moiety, or a linker attached thereto is derivatized as a methanethiosulfonate which can then be reacted with the cysteines -SH group as described herein.
- Detailed protocols for the preparation of mutant enzymes and the coupling of a targeting moiety are provided below and in the examples.
- any residue of the enzyme can be selected for mutagenesis (e.g. substitution of a cysteine) and chemical modification to introduce a targeting moiety, as long as the modification retains the desired level of activity of the subject enzyme.
- mutagenesis e.g. substitution of a cysteine
- chemical modification to introduce a targeting moiety, as long as the modification retains the desired level of activity of the subject enzyme.
- this is accomplished by making the substitution at a location that does not block critical substrate interactions or drastically alter folding/conformation of the subject enzyme.
- Suitable sites for introduction of a targeting moiety can be determined by substituting cysteine, and optionally an attached targeting moiety, and assaying the enzymes for the desired activity. With the cu ⁇ ent advances in combinatorial chemistry and high throughput screening systems such modifications and screening can be accomplished with only routine experimentation.
- prefe ⁇ ed sites include sites not in critical conformation determining regions and sites disposed away from the subsite(s) of the enzyme.
- prefe ⁇ ed amino acid residues selected for modification include residues expected to be important discriminatory sites near, adjacent to or within the substrate binding region of the enzyme. Such residues are determined from mutagenesis experiments where the subsite residues are systematically mutagenized and the effect of such mutagenesis on binding specificity and/or enzymatic activity is determined.
- important residues can be identified from inspection of crystal structures of the enzyme alone or in complex with subtrate, substrate analogues or inhibitors and/or from predicted protein folding or protein-protein interactions determined using protein modeling software (e.g., Quanta, Cerius, Insight (Molecular Simulations Inc.) and Frodo (academic software).
- Side chains situated to alter interaction at subsites defined by Berger and Schechter can be selected based on the crystallographic models of the enzymes and extrapolated to homologous enzymes if necessary if structural information on a specific enzyme is unavailable.
- R. lentus subtilisin sites 62, 156, 166, 217 and 222 are important substrate specificity determining sites.
- Additional related sites include position 96, 104, 107, 189 and 209 in subtilisin and homologous positions in related enzymes.
- residues typically lie in the SI, S2, S4, SI', S2', or S3' subsites although it will be appreciated that in certain cases, alteration of residues in other subsites can also produce dramatic effects.
- prefe ⁇ ed residues for mutation include, but are not limited to residues at or near residues 156 and 166 in the SI subsite, residues 217 and 222 in the SI' subsite, residue 62 in the S2 subsite, and Leu96, Val 104, He 107, Phe 189 and Tyr209 or residues at or near homologous positions other subtilisin-type serine proteases (preferably positions within subsites).
- prefe ⁇ ed residues for mutation include, but are not limited to, residues at or near residues Tyr94, Leu99, Glnl75, Aspl89, Serl90, Glnl92, Leul 11, Phel75, Tyrl76, Serl82, Leul84, Phel89, Tyr214, Asp231, Lys234, and Ile243 of trypsin (Protein Databank Entry 1TPP) or residues at or near homologous positions of other chymotrypsin-type (trypsin-chymotrypsin-type) serine proteases (preferably positions within subsites).
- prefe ⁇ ed residues for mutation include, but are not limited to, residues at or near the following residues: Trpl04, Thrl38, Leul44, Vall54, Ilel89, Ala 225, Leu278 and Ilel85, where these are residues o ⁇ Candida antartica lipase (Protein Data Bank entry ltca) or residues at homologous positions of other alpha/beta type serine hydrolases (preferably positions within subsites).
- amino acids replaced in the enzyme by cysteines are selected from the group consisting of asparagine, leucine, methionine, or serine. More preferably the amino acid to be replaced is located in or near a subsite of the enzyme preferably the SI, SI' or S2 subsites.
- subtilisin the amino acids to be replaced are N62, L217, M222, S156, S166, site 104, site 107 (S4), site 96 (S2), site 189(S2'), and site 209 (SI 7S3') or their homologues where the numbered position co ⁇ esponds to naturally occurring subtilisin from Bacilus amyloliquefaciens or to equivalent amino acid residues in other subtilisins such as Bacillus lentus subtilisin.
- the chimeric molecules of this invention are not limited to serine hydrolases.
- this invention includes other chimeric proteases.
- proteases include, but are not limited to aspartyl proteases, cysteine proteases, metalloproteases, and the like.
- prefe ⁇ ed residues for mutation include, but are not limited to, amino acid(s) co ⁇ esponding (e.g. at a homologous position) to a residue at or near the following residues Tyr9, Metl2, Glnl3, Gly76, Thr77,
- protease is cysteine protease
- prefe ⁇ ed residues for mutation include, but are not limited to, amino acid(s) co ⁇ esponding (e.g. at a homologous position) to a residue at or near the following residues Asnl ⁇ , Ser21, Asn64, Tyr67, Trp69, Glnl 12, Glnl42, Aspl58, Trpl77, and Phe207, where these reference residues are residues in the mature papain (Protein Data Bank entry 1BQI).
- prefe ⁇ ed residues for mutation include, but are not limited to, amino acid(s) co ⁇ esponding (e.g. at a homologous position) to a residue at or near the following residues Leul 11, Phe 175, Tyr 176, Ser 182, Leu 184, Phel89, Tyr214, Asp231, Lys234, and Ile243, where these reference residues are residues in the mature human matrix metalloprotease (Protein Data Bank entry 830C).
- the mutants described herein are most efficiently prepared by site-directed mutagenesis of the DNA encoding the wild-type enzyme of interest (e.g. Bacillus lentis subtilisin). Techniques for performing site-directed mutagenesis or non-random mutagenesis are known in the art. Such methods include, but are not limited to alanine scanning mutagenesis (Cunningham and Wells (1989) Science, 244, 1081-1085), oligonucleotide-mediated mutagenesis (Adellman et al. (1983) DNA, 2, 183), cassette mutagenesis (Wells et al. ( 1985) Gene, 344: 315) and binding mutagenesis (Ladner et al. WO 88/06630).
- the substitute amino acid residue (e.g. cysteine) is introduced into the selected position by oligonucleotide-mediated mutagenesis using the polymerase chain reaction technique.
- the gene encoding the desired native enzyme (e.g. subtilisin) is carried by a suitable plasmid.
- the plasmid is an expression vector, e.g., a plasmid from the pBR, pUC, pUB, pET or pHY4 series.
- the plasmid can be chosen by persons skilled in the art for convenience or as desired.
- the fragment containing the selected mutation site is cleaved from the gene encoding the subject enzyme by restriction endonucleases and is used as the template in a modified PCR technique (see, Higuchi et al. (1988) Nucleic Acid Res., 16, 7351-7367).
- a modified PCR technique see, Higuchi et al. (1988) Nucleic Acid Res., 16, 7351-7367.
- an oligonucleotide containing the desired mutation is used as a mismatch primer to initiate chain extension between 5' and 3 PCR flanking primers.
- the process includes two PCR reactions. In the first PCR, the mismatch primer and the 5' primer are used to generate a DNA fragment containing the desired base substitution. The fragment is separated from the primers by electrophoresis. After purification, it is then used as the new 5' primer in a second PCR with the 3' primer to generate the complete fragment containing the desired base substitution. After confirmation of the mutation by sequencing, the mutant fragment is then
- a cassette mutagenesis method may be used to facilitate the construction and identification of the cysteine mutants of the present invention.
- the gene encoding the serine hydrolase is obtained and sequenced in whole or in part.
- the point(s) at which it is desired to make a mutation of one or more amino acids in the expressed enzymes is identified.
- the sequences flanking these points are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide which when expressed will encode the desired mutants.
- restriction sites are preferably unique sites within the serine hydrolase gene so as to facilitate the replacement of the gene segment.
- any convenient restriction site which is not overly redundant in the hydrolase gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (e.g., from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such a fashion that neither the reading frame nor the amino acids encoded are changed in the final construction.
- the task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the genetic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. If convenient flanking restriction site is available, the above method need be used only in connection with the flanking region which does not contain a site.
- Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished e.g., M13 primer extension in accord with generally known methods.
- the restriction sites flanking the sequence to be mutated are digested with the cognate restriction enzymes and the end termini-complementary oligonucleotide cassette(s) are ligated into the gene.
- the mutagenesis is enormously simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites.
- a suitable DNA sequence computer search program simplifies the task of finding potential 5' and 3' convenient flanking sites.
- any mutation introduced in creation of the restriction site(s) are silent to the final construction amino acid coding sequence.
- a candidate restriction site 5' to the target codon a sequence preferably exists in the gene that contains at least all the nucleotides but for one in the recognition sequence 5' to the cut of the candidate enzyme.
- the blunt cutting enzyme Smal CCC/GGG
- N needs to be altered to C this alteration preferably leaves the amino acid coding sequence intact.
- a particularly prefe ⁇ ed of method of introducing cysteine mutants into the enzyme of interest is illustrated with respect to the subtilisin gene from Bacillus lentus ("SBL").
- the gene for SBL is cloned into a bacteriophage vector (e.g. M13mpl9 vector) for mutagenesis (see, e.g. U.S. Patent 5,185,258).
- Oligonucleotide- directed mutagenesis is performed according to the method described by Zoller et al. (1983) Meth. Enzymol, 100: 468-500.
- the mutated sequence is then cloned, excised, and reintroduced into an expression plasmid (e.g. plasmid GG274) in the B. subtihs host.
- PEG 50%) is added as a stabilizer.
- the crude protein concentrate thus obtained is purified by first passing through a SephadexTM G-25 desalting matrix with a pH 5.2 buffer (e.g. 20 mM sodium acetate, 5 mM CaCl 2 ) to remove small molecular weight contaminants. Pooled fractions from the desalting column are then applied to a strong cation exchange column (e.g.
- SP SepharoseTM FF in the sodium acetate buffer described above and the SBL is eluted with a one step gradient of 0-200 mM NaCl acetate buffer, pH 5.2.
- Salt-free enzyme powder is obtained following dialysis of the eluent against Millipore purified water and subsequent lyophilization.
- the purity of the mutant and wild-type enzymes, which are denatured by incubation with a 0J M HC1 at 0°C for 30 minutes is ascertained by SDS-PAGE on homogeneous gels (e.g. using the PhastTM system from Pharmacia, Uppsala, Sweden).
- the concentration of SBL is determined using the Bio-Rad (Hercules, CA) dye reagent kit which is based on the method of Bradford (1976) Anal. Biochem., 72: 248-254). Specific activity of the enzymes is determined as described below and in the examples.
- kits for site-directed mutagenesis are commercially available (see, e.g. TransfomerTM Site-Directed Mutagenesis Kit available from Toyobo).
- chemical coupling of the targeting moiety is to a cysteine, either naturally occurring in the subject enzyme or introduced (e.g. via site-directed mutagenesis.
- the chimeric molecules of this invention need not be limited to molecules conjugated through cysteines. In certain embodiments the conjugation can be through virtually any other amino acid (e.g., a serine, a glycine, a tyrosine, etc.).
- the conjugation can be through the existing R group (using other coupling chemistries), or alternatively a sulfhydryl group (SH) can be introduced (linked) to the R group and the targeting moiety, derivatized as a methanethiosulfonate reagent, can be coupled, e.g. as illustrated in the examples.
- a sulfhydryl group SH
- the targeting moiety derivatized as a methanethiosulfonate reagent
- the mutated enzyme is expressed from a heterologous nucleic acid in a host cell.
- the expressed enzyme is then isolated and, if necessary, purified.
- the choice of host cell and expression vectors will to a large extent depend upon the enzyme of choice and its source.
- a useful expression vector contains an element that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in a host cell independent of the genome of the host cell, and preferably one or more phenotypic markers that permit easy selection of transformed host cells.
- the expression vector may also include control sequences encoding a promoter, ribosome binding site, translation initiation signal, and, optionally, a repressor gene, a selectable marker or various activator genes.
- nucleotides encoding a signal sequence may be inserted prior to the coding sequence of the gene.
- a gene or cDNA encoding a mutated enzyme to be used according to the invention is operably linked to the control sequences in the proper reading frame.
- Suitable host cells include bacteria such as E. coli or Bacillus, yeast such as S. cerevisiae, mammalian cells such as mouse fibroblast cell, or insect cells.
- a bacterial expression system is used.
- the host is Bacillus.
- Protein expression is performed by processes well known in the art according to factors such as the selected host cell and the expression vector to culture the transformed host cell under conditions favorable for a high-level expression of the foreign plasmid. Methods of cloning and expression of peptides are well known to those of skill in the art. See, for example, Sambrook, et al.
- one particularly prefe ⁇ ed expression system is plasmid GG274 which is then expressed in a B. subtilis host.
- targeting moieties can be coupled to the cysteine(s) introduced into the subject enzyme (e.g. serine hydrolase).
- the targeting moiety is selected depending on the desired use of the enzyme.
- suitable targeting moieties include, but are not limited to, moieties that are bound by receptors, targeting moieties that are bound by antibodies and enzymes, targeting moieties that are bound by lectins, and various other targeting moieties.
- the targeting moieties are drugs or prodrugs that are specifically bound by a receptor and/or an enzyme.
- the R group on cysteines provides a convenient relatively reactive thiol group (-SH) that can be exploited for coupling a desired targeting moiety to the cysteine.
- the targeting moiety of interest is provided, derivatized as a methanethiosulfonate reagent which, when reacted with the cysteine, results in the substituent of interest covalently coupled to the cysteine by a disulfide linkage (-S-S-).
- Reaction mixtures are kept at 20°C with continuous end-over-end mixing. Reactions are monitored by following the specific activity (e.g. with suc-AAPF-pNA) and by tests for residual free thiol (e.g. with Ellman's reagent).
- the reaction mixture is loaded on a SephadexTM PD-10 G25 column with 5 mM MES and 2 mM CaC12, pH 6.5.
- the protein fraction is then dialyzed against 1 mM CaC12 and the dialysate is lyophilized. In a particulary prefe ⁇ ed embodiment the fraction is dialyzed against pH 6.5 MES then flash frozen.
- the reactive groups may be derivatized with appropriate blocking/protecting groups to prevent undesired reactions during the coupling.
- the serine hydrolase contains one or more cysteines that are not to be derivatized
- the cysteines may be replaced with other amino acids (e.g. via site directed mutagenesis) and/or the thiol group(s) on these cysteines may be derivatized with appropriate protecting groups (e.g. (e.g. benzyl, trityl, tert-butyl, MOM, acetyl, thiocarbonate, thiocarbamate, and others).
- blocking/protecting groups are well know to those of skill in the art (see, e.g., Protective Groups in Organic Synthesis” Theodora W. Greene and Peter G. M. Wuts Third Edition, Wiley Jnterscience, Toronto, (1999), pp 454-493.) While in particularly prefe ⁇ ed embodiments, a cysteine is introduced/substituted into the enzyme, in certain embodiments, other amino acids (e.g. lysine, histadine, etc.) may be introduced, and in certain embodiments, the targeting moiety may be coupled to these residues.
- other amino acids e.g. lysine, histadine, etc.
- the chimeric molecules of this invention are typically screened for the activity or activities of interest.
- the activity of interest depends on the desired use of the chimeric molecule.
- the chimeric molecule may be assayed for two properties: 1) The ability to reduce or eliminate the activity of the target, e.g., where the target is biologically active, or simply to partially or fully degrade the target, e.g. where the target is not biologically active; and 2) the ability to release from the target after the target is degraded and to bind and degrade another target.
- the chimieric molecule may simply be assayed for activity (e.g. the ability to perform degradations) in a substoichiometric manner.
- activity of a molecule on a cell surface receptor can be determined by providing a cell expressing the receptor and measuring the activity of the receptor in the presence or absence of the chimeric molecule.
- Receptor assays are commonly performed in oocytes (e.g. Xenopus oocytes) into which an RNA encoding the subject receptor is inserted. Receptor activity is monitored by measuring electrochemical activity (e.g.
- V Illustrative uses of catalytic antagonists and/or redirected enzymes.
- the catalytic antagonists of this invention can be used as therapeutic in a wide number of pathologies including, but not limited to inhibitors of viral infection and/or replication, inhibitors of bacterial infection and/or biofilm formation, modulators of an immune response, modulators of an autoimmune response, inhibitors of an inflammatory response, and the like. More generally, as indicated above, the catalytic antagonists of this invention can be used to replace existing pharmaceuticals where the pharmaceutical acts by inhibiting and/or antagonizing a receptor.
- a receptor bound by a catalytic antagonist of this invention is degraded (thereby releasing the catalytic antagonist to act on another receptor). Because the receptor is degraded it does not recover its activity.
- the catalytic antagonists are thus expected to provide greater efficacy at a lower dosage and to provide longer lasting activity at a particular dosage.
- this invention provides methods of improving the activity of a drug.
- the methods involve attaching the drug to an enzyme capable of degrading the target (e.g. receptor) to which the drug binds.
- an enzyme capable of degrading the target e.g. receptor
- Prefe ⁇ ed enzymes in this context include hydrolases and even more preferably include proteases (e.g. serine proteases, metalloproteases, cysteine proteases, aspartyl proteases, etc.).
- the targeting moieties need not be limited to drugs.
- a wide variety of other targeting moieties are suitable as well and provide catalytic antagonists useful in a wide variety of therapeutic contexts.
- the targeting moiety can be a molecule that specifically binds to the CCR5 and/or CXCR2 receptors, commonly found on lymphocytes (e.g. T-cells).
- CCR5 and CXCR2 receptors are implicated in the infection of a cell by HIV and persons defective in one or more of these receptors typically demonstrate resistance to HIV infection.
- Targeted destruction/inhibition of either or both of these receptors e.g. by a catalytic antagonist comprising a CCR5 and/or CXCR2 specific targeting agent attached to a suitable hydrolase (e.g. subtilisin) will increase the target cell's resistance to HIV infection.
- a catalytic antagonist comprising a CCR5 and/or CXCR2 specific targeting agent attached to a suitable hydrolase (e.g. subtilisin) will increase the
- glycosidases involved in N-linked protein glycosylation can be specifically targeted (e.g. using an Aza-sugar targeting moiety) attached to a suitable hydrolase (e.g. subtilisin, pepsin, etc.).
- a catalytic antagonist will be of use in the treatment of HIV, herpes, other viruses, and various cancers.
- Catalytic antagonists comprising a sugar targeting moiety attached to a suitable hydrolase (e.g. subtilisin, pepsin, etc.) will be useful in the treatment of a wide variety of conditions, including inflammatory responses (e.g. associated with arthritis, septic shock, myocardial infarction, etc.), bacterial binding to cells and subsequent infection (e.g. H. pylori infection associated with ulcers).
- a suitable hydrolase e.g. subtilisin, pepsin, etc.
- bacterial binding to cells e.g. H. pylori infection associated with ulcers
- the targeting moiety is a Gal ⁇ (l,4)Gal pathogenic E. coli infections can be blocked.
- lectin-directed catalytic antagonists can be used to inhibit biofilm formation in vivo or ex vivo.
- the targeting moiety can be a sialic acid sugar (or mimetics such as Rilenza (Glaxo- Wellcome) or the like).
- a suitable hydrolase e.g. subtilisin
- the catalytic antagonist can specifically target the sialidase activity of viruses such as influenza.
- the chimeric catalytic antagonists of this invention can also be used to specifically target hyperproliferative and/or invasive cells (e.g. metastatic cells).
- Various enzymes e.g. especially matrix metalloproteases
- these enzymes can be specifically targeted using a number of targeting moieties (e.g. crown ethers).
- suitable hydrolase e.g. a subtilisin
- the catalytic antagonist will degrade the target enzyme (e.g. metalloprotease) and thereby interfere with or eliminate the ability of a cell to invade, e.g. a basement membrane, thereby slowing the progression of a cancer.
- the catalytic antagonists of this invention can also be used to target and alter a variety of immune processes.
- alpha-Gal epitope disaccharide (Gal ⁇ (l,3)Gal) can be used as a targeting moiety and (e.g. when attached to a subtilisin) can bind to and specifically inhibit alpha-Gal epitope specific antibodies thereby mitigating host rejection of a xenograft.
- known allergens e.g. various pollens or epitopes present on such allergens
- the chimeric molecules can be used in various drug delivery strategies to specifically target a therapeutic activity to a cell, organ, or tissue of interest.
- enzymes capable of converting prodrugs to their active form are attached to a targeting moiety (e.g. a tumor cell specific moiety) that localizes them to the site of desired activity (e.g. a tumor cell)
- a targeting moiety e.g. a tumor cell specific moiety
- various prodrugs are known to those of skill in the art and include, but are not limited to ganclovir activated by thymidine kinase, phosphenytoin converted to phenytoin by a phosphatase, depivefrin is converted to epinephrine by an esterase and the like.
- glucocerebrosidase may be directed, e.g. to spleen cells in the treatment of Gaucher's or Tay-Sachs disease.
- superoxide dismutase may be attached to liver cell specific targeting moieties so that it is targeted to liver tissue where it can provide anti-oxidant activity.
- the chimeric molecules of this invention can be used to target and destroy particular preselected molecules whether or not they have a biological activity.
- components of various soils or stains e.g. milk, blood, eggs, grass stains, oil stains, etc.
- avidin/egg protein can be specifically targeted by using a biotin as a targeting moiety to specifically directed, e.g. a protease to the site. The stain is degraded/digested and thereby released from the underlying substrate.
- Such specifically targeted chimeras are particularly in various cleaning formulations.
- the therapeutic chimeric molecules of this invention are useful for intravenous, parenteral, topical, oral, or local administration (e.g., by aerosol or transdermally).
- Particularly prefe ⁇ ed modes of administration include intra-arterial injection, more preferably intra-peritoneal intra-hepatic artery injection or, where it is desired to deliver a composition to the brain, (e.g., for treatment of brain tumors) a carotid artery or an artery of the carotid system of arteries (e.g., occipital artery, auricular artery, temporal artery, cerebral artery, maxillary artery, etc.).
- the chimeric molecules are typically combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition.
- Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts, for example, to stabilize the composition or to increase or decrease the absorption of the agent.
- Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the anti-mitotic agents, or excipients or other stabilizers and/or buffers.
- physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms.
- Various preservatives are well known and include, for example, phenol and ascorbic acid.
- a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the route of administration of the chimeric molecule and on the particular physio-chemical characteristics of the agent.
- the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
- unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges. It is recognized that the chimeric molecules, when administered orally, are preferably protected from digestion.
- compositions are either delivered directly to the desired site (e.g. by injection, cannulization, or direct application during a surgical procedure) or they are solubilized in an acceptable excipient.
- compositions of this invention are useful for topical administration e.g., in surgical wounds to treat incipient tumors, neoplastic and metastatic cells and their precursors.
- the compositions are useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ.
- the compositions for administration will commonly comprise a solution of the chimeric molecule agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier for water-soluble chimeric molecules.
- a pharmaceutically acceptable carrier preferably an aqueous carrier for water-soluble chimeric molecules.
- a variety of carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
- These compositions may be sterilized by conventional, well known sterilization techniques.
- the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate,
- the concentration of chimeric molecule in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania (1980). Dosages for typical chemotherapeutics are well known to those of skill in the art. Moreover, such dosages are typically advisorial in nature and may be adjusted depending on the particular therapeutic context, patient tolerance, etc. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient.
- the composition should provide a sufficient quantity of the proteins of this invention to effectively treat the patient.
- dosage for a typical pharmaceutical composition for intravenous administration would be about 0.01 to per patient per day. Dosages from 0J up to about 1000 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ.
- kits for the creation and/or use of the chimeric molecules of this invention comprise one or more containers containing one or more targeting moieties derivatized as methanesulfonates for coupling to a cysteine in an enzyme.
- the kits may comprise one or more enzymes, more preferably mutant enzymes having an inserted cysteine ready for coupling to a methanesulfonate derivatized targeting moiety. When provided in this manner the kits enable one or ordinary skill in the art to assemble the desired chimeric molecule for a particular use.
- one typically kit may include a multiplicity of methanesulfonate derivatized targeting moieties and one or more enzymes suitable for coupling.
- the desired enzyme is then reacted (as described herein) with the desired targeting moiety to produce the desired chimeric molecule.
- kits may additional comprise one or more of the reagents utilized in a typical coupling reaction.
- this invention provides one or more chimeric molecules (e.g. catalytic antagonists and/or redirected enzymes) of this invention.
- the chimeric molecules can be provided as a dry (e.g. lyophilized powder) or in solution and/or as an emulsion.
- the chimeric molecules are provided in, or along with, a pharmacological excipient and, optionally, may be provided in a unit dosage format.
- the kits may optionally include any reagents and/or apparatus to facilitate the uses described herein. Such reagents include, but are not limited to buffers, organic solvents, labels, labeled antibodies, bioreactors, cells, etc.
- kits may include instructional materials containing directions (i.e., protocols) for the assembly of chimeric molecules of this invention and/or for the use thereof.
- instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
- Such media may include addresses to internet sites that provide such instructional materials.
- Examples 1-4 demonstrates the highly specific selectivity of a catalytic antagonist of this invention in which the targeting moiety is a known enzyme inhibitor.
- Examples 5 through 7 detail the construction and evaluation of chimeric molecules in which the chimeric molecules are targeted to the binding protein lectin concanavalin A.
- Examples 8 through 10 detail the construction and evaluation of chimeric molecules in which the chimeric molecules are targeted to the binding protein avidin.
- Examples 11 and 12 detail the construction and evaluation of chimeric molecules in which the chimeric molecules are targeted to a monoclonal anti-biotin antibody IgG.
- Example 13 details the respective stoichiometry of these examples.
- Example 1 Targeting Enzymes with Inhibitors Synthesis and Attachment of an HLADH inhibitor to SBL and Characterization of the Resulting SBL-S-pyrazole CMMs
- the inhibitor(s) chosen as targeting moieties for this approach are strong inhibitor(s)/degraders of the target enzyme, but are poor inhibitors of the CMM.
- alcohol dehydrogenase (ADH) which is strongly inhibited by 4-pyrazole derivatives, was chosen as the target enzyme and the inhibitors chosen as targeting were pyrazoles known to inhibit ADH.
- the modified CMM in this case was a subtilisin (SBL).s
- HLADH horse liver alcohol dehydrogenase
- N62C, L217C, S166C, and S156C mutants were modified with the MTS- pyrazole reagent 4 by reaction at pH 9.5 following the standard protocol. In all cases the resulting enzymes were active after modification and the data for amidase kinetics (substrate suc-AAPFpNA) and ESMS are shown in Table 2.
- the S166C- S-Pyrazole CMM shows the lowest k c KM, about 9 times smaller than for SBL-WT.
- the kcat of the SI 56- and L217C-S-Pyrazole CMM were both very similar and about 2.5 times smaller than for the WT enzyme. .Their substrate binding properties, however, were fairly different: S156C-S-Pyrazole bound better than SBL-WT whereas the K M of L217C-S-Pyrazole is larger than that of the WT enzyme. N62C-S-Pyrazole is slightly more active than the other pyrazole CMMs and its k cat is just 1.5 times smaller compared to SBL-WT. However it had the largest K M among all pyrazole-CMMs and its k c KM was about 2 times smaller compared to SBL-WT.
- the reaction was poured onto a pre-packed, pre-equilibrated G-25 Sephadex ® PD10 column and eluted with 3.5 mL Quench Buffer (see below).
- the eluant was dialysed at 4° C against 10 mM MES, 1 mM CaCl 2 pH 5.8 (2 _ IL, 2 _ 45 min).
- the resulting dialysate was flash frozen in liquid nitrogen and stored at -18° C.
- Modifying Buffer pH 9.5 140 mM CHES, 2 mM CaCl 2 pH 7.5 140 mM HEPES, 2 mM CaCl 2 pH 5.5 140 mM MES, 2 mM CaCl 2 Quench Buffer: Reactions at pH 7.5 - 9.5: 5 mM MES 1 mM CaCl 2 pH 6.5 Reactions at pH 5.5: 5 mM MES 1 mM CaCl 2 pH 5.5
- CMMs Prior to ES-MS analysis CMMs were purified by FPLC (BioRad, Biologic System) on a Source 15 RPC matrix (17-0727-20 from Pharmacia) with 5% acetonitrile, 0.01% TFA as the running buffer and eluted with 80% acetonitrile, 0.01% TFA in a one step gradient.
- FPLC BioRad, Biologic System
- Michaelis-Menten constants were measured at 25 °C by curve fitting (Grafit 3.03) of the initial rate data determined at eight concentrations (31.25 ⁇ M - 2.0 mM) of the succinyl-AAPF-SBn substrate, followed indirectly using Ellman's reagent in OJ M Tris.HCl, containing 0.005 vol% Tween-80, 1 vol% 37.5 mM Ellman's reagent in DMSO, pH 8.6.
- Targeted association of CMMs with HLADH via the pyrazole inhibitor should lead to selective hydrolysis. If hydrolysis of the HLADH takes place, the oxidoreductase activity of the HLADH should be diminished or eradicated after a certain time of incubation with our CMMs. To demonstrate this, the "Targeting Assay" as described above was carried out again after 4h incubation. Remaining HLADH activity was determined by addition of cyclohexanol as substrate. The results are shown in Figure 5.
- a Assay buffer 0J M Glycine-NaOH, pH 9.0.
- b Solution (10 mg/mL) in Assay buffer.
- c Solution (33.2 mg/mL) in Assay buffer.
- d Solution (10 mg/mL, 52.4% activity) in TRIS-HC1 buffer (0.05M TRIS, pH 7.4).
- e The amounts are calculated for equal concentrations of active enzyme (as determined by initial rate kinetics with succ-AAPFpNA).
- subtilisins e.g. SBL
- CMM site directed mutagenesis chemical modification
- HLADH activity was monitored by periodically withdrawing a portion of the digestion mixture, and assessing the ability of the aliquot to oxidize cyclohexanol to cyclohexanone.
- the reaction course was monitored by observing the change of NAD + to NADH at 340 nm as cyclohexanol was oxidized.
- Alkaline phosphatase (AP) activity was monitored by periodically withdrawing a portion of the digestion mixture, and assessing the ability of the aliquot to hydro lyzep-nitrophenyl phosphate to inorganic phosphate and -nitrophenolate. The reaction course was monitored by observing the appearance of 7-nitrophenolate at 405 nm.
- HLADH activity was assessed by monitoring the NAD to NADH conversion at 340 nm as cyclohexanol was oxidized (see experimental). Incubation at 35 °C. Table 8. Alkaline phosphatase activity after incubation relative to initial value.
- Triethanolamine-HCl buffer with 3 M NaCl 0.1 mM Mg 2+ and 0.01 mM Zn 2+ (pH 7.8 buffer): Triethanolamine (0.05 moles, 7.5 g), NaCl (3 moles, 175.5 g), MgC12 (0J mL of 1 M solution) and ZnC12 (0J mL of IM solution) were dissolved in MQ water (ca. 900 mL). The pH was adjusted to 1.4 with ca 2N HCl and the resulting solution made up to IL.
- PH 8.6 ca. 0.1 M TRIS-HC1 buffer with 0.05% Tween. 1 mM Mg 2+ and 0.1 mM Zn 2+ (pH 8.6 buffer):
- MgCl 2 (0J mL of a 1 M solution in MQ water) and ZnCl 2 (0J mL of a 0.1 M solution in MQ water) were added to a 100 mL volumetric flask, and the flask was made up to the mark with pH 8.6 0J M TRIS-HC1 buffer containing 0.05% Tween.
- HLADH solution Horse liver alcohol dehydrogenase (Sigma A-9589, EC 1JJJ, 8 mg of ca.
- Alkaline phosphatase (Boehringer Mannheim 713 023, EC 3J.3J, ca. 950 ⁇ L as received in 50% w/v glycerol: buffer) was diluted with pH 7.8 buffer (ca. 15 mL), and was concentrated at 4 °C to 10-20% of its original volume using a Centriprep concentrator. A further 15 mL of pH 7.8 buffer was added, and the sample was concentrated once more. This process was repeated a further 3 times using pH 9.0 assay buffer for dilutions. After the third concentration the concentrate (ca. 1.85 mL) was collected and was stored on ice. This procedure was necessary to remove glycerol, which is a substrate for HLADH.
- NAD + solution 33.2 mg/mL of NAD + was dissolved in pH 9.0 assay buffer.
- PNPP solution p-Nitrophenyl phosphate solution
- CMM was S166C-pyrazole.
- the vials were incubated at 35 °C for the times indicated in the tables below. Aliquots were periodically withdrawn in order to assay the HLADH and alkaline phosphatase activities as time progressed.
- a portion of solution (65 ⁇ L) was withdrawn from an incubation vial and was then injected into a micro-cuvette containing pH 9.0 assay buffer (200 ⁇ L). The cuvette was incubated at 25 °C for 2 minutes, and then cyclohexanol solution (30 ⁇ L) was added. The absorbance at 340 nm was then monitored for 300 s, and the O.D. change per second up to 0.2 absorbance units was recorded.
- Assaying Alkaline Phosphatase Activity A portion (20 ⁇ L) was withdrawn from an incubation vial and was then injected into pH 8.6 buffer (980 ⁇ L). The mixture was vortexed. 10 ⁇ L was the removed from the mixture, and was injected into a cuvette containing 990 ⁇ L of PNPP solution incubated at 25 °C. The absorbance change at 405 nm was monitored for 150 s, and the O.D. change per second up to 1 Absorbance unit was recorded.
- HLADH (ca. 79 kD for the dimer)
- SBL or pyrazole-CMM (ca. 27 kD)
- HLADH solutions were incubated in the presence of WT-SBL, S166C- pyrazole or S156C-pyrazole.
- a control experiment was performed in the absence of any SBL-based enzyme (HLADH alone).
- the HLADH activities of the four mixtures were periodically assayed (see experimental) — see Table 11.
- HLADH activity was assessed by monitoring the conversion of NAD + to NADH at 340 nm as cyclohexanol was oxidized at 25 °C, pH 9.0 (see experimental).
- CMMs reflects the ability of the CMMs to target and thus inhibit HLADH.
- S156C-pyrazole and, to a lesser extent, S166C-pyrazole clearly cause dramatic reductions in HLADH activity on incubation.
- the pyrazole-CMMs were used in less than stoichiometric amounts with respect to HLADH — 4 eq. HLADH active sites: 1 eq. pyrazole-CMM — but they rapidly caused a greater than 25% diminution of HLADH activity.
- HLADH activity is seen to drop from 67% to 5% over 20 h, representing a 13.5- fold reduction of HLADH activity over-and-above the maximum inhibitory effect of the pyrazole moiety.
- WT-SBL causes a mere 1.4-fold reduction of HLADH activity over the same 20 h period, despite its enhanced amidase specific activity when compared to the pyrazole-CMMs.
- Glycine (OJ mol) was dissolved in water (ca. 800 mL). A solution of Tween 80 (50 mL of a 0.1% v/v in MQ water) was added, and the pH was adjusted to 9.0 with ca. 5 M NaOH solution. The mixture was made up to IL with MQ water.
- TRIS (302.9 mg, 2.5 mmol) was dissolved in MQ water (ca. 40 mL). The pH was adjusted to 1.4 with ca. 1 M HCl solution, and the volume of the mixture was made up to 50 mL with MQ water.
- Horse liver alcohol dehydrogenase (Sigma A-9589, Lot 58H7004, EC 1.1.1.1, 8.45 mg of 52.4 % w/w protein — according to manufacturer's Biuret titration) was dissolved in pH 1.4 TRIS (0.845 mL) to give a 5.24 mg/mL solution of active protein.
- HLADH (50 ⁇ L) solution was added to pH 1.4 TRIS (450 ⁇ L) to give a tenfold diluted solution.
- Bradford (Bio-Rad) protein determination was performed on this diluted sample, and yielded a protein concentration of 0.616 mg/mL. This translates to a concentration of 6J6 mg/mL in the original HLADH stock.
- NAD + (332 mg) was dissolved in pH 9.0 assay buffer (10 mL) to give a 33.2 mg/mL solution.
- WT-SBL (1.88 mg of dry powder, 73% w/w active protein) was dissolved in pH 5.8, 10 mM MES, 2 mM CaCl 2 "storage buffer" (500 ⁇ L) to give a 2.74 mg/mL solution of active WT-SBL.
- S156C-pyrazole and S166C-pyrazole were previously titrated with PMSF: their concentrations were 2.5 mg/mL and 3.62 mg/mL respectively.
- WT-SBL 2.74 mg/mL; S166C-pyrazole, 3.62 mg/mL; S156C-pyrazole, 2.5 mg/mL.
- the tubes were kept on ice until the HLADH activity of each tube had been assayed in order to give a "time zero" value for each tube (see below for assay protocol).
- the tubes were then incubated on a water bath at 35°C. Periodically, 700 ⁇ L of reaction mixture with withdrawn from each falcon tube, the aliquots were placed in individual eppendorf tubes, and the eppendorf tubes were stored on ice. The content of each eppendorf tube was then assayed for HLADH activity (see below).
- reaction mixture 650 ⁇ L was injected into a cuvette containing pH 9.0 assay buffer (2.00 mL). The cuvette was incubated at 25 °C for 2 minutes, and then cyclohexanol solution (300 ⁇ L) was added. After a 10 s delay, the absorbance at 340 nm was monitored for 300 s. Tthe O.D. change per second up to 0.2 absorbance units was used to calculate an initial rate.
- HLADH (ca. 79 kD for the (AP) WT-SBL or Pyrazole-CMM dimer) (ca. 134 kD for each (ca. 27 kD) dimer)
- each vial contained one of: buffer (no SBL added), WT-SBL, S156C-pyrazole or S166C-pyrazole.
- Alkaline phosphatase is clearly not very susceptible to hydrolysis by WT-SBL or Pyrazole-CMMs. HLADH activity is not significantly diminished on incubation in the absence of SBL or in the presence of WT-SBL. However, in the presence of S 156C-pyrazole or S166C-pyrazole HLADH activity is seen to diminish rapidly.
- Pyrazole-CMMs are seen to target and to catalytically destroy HLADH in the presence of alkaline phosphatase.
- Alkaline phosphatase is unaffected by the hydrolytic action of WT-SBL and Pyrazole-CMMs.
- Glycine (0J mol) was dissolved in water (ca. 800 mL).
- Magnesium chloride solution (1 mL of a 1 M solution) and zinc chloride solution (1 mL of a 0J M solution) were added to the glycine solution, and the of the mixture pH was adjusted to 9.0 with ca. 5 M NaOH solution.
- the mixture was made up to IL with MQ water.
- TRIS (302.9 mg, 2.5 mmol) was dissolved in MQ water (ca. 40 mL). The pH was adjusted to 1.4 with ca. 1 M HCl solution, and the volume of the mixture was made up to 50 mL with MQ water.
- TRIS (6.057 g, 0.05 mol) was dissolved in MQ water (ca. 800 mL).
- MQ water ca. 800 mL
- Magnesium chloride solution (1 mL of a 1 M solution) and zinc chloride solution (1 mL of a 0J M solution) were added to the TRIS solution, and the of the mixture pH was adjusted to 7.4 with ca. 1 M HCl solution.
- the mixture was made up to IL with MQ water.
- 1 mM Me2+ and 0.1 mM Zn2+(pH 8.6 buffer) MgCl 2 (0J mL of a 1 M solution in MQ water) and ZnCl 2 (0J mL of a 0J M solution in MQ water) were added to a 100 mL volumetric flask, and the flask was made up to the mark with pH 8.6 0J M TRIS-HCl buffer containing 0.05% Tween (standard amidase kinetics buffer).
- NAD + 39.15 mg was dissolved in pH 9.0 assay buffer (1J79 mL) to give a 33.2 mg/mL solution.
- Cyclohexanol solution Cyclohexanol (100 mg) was dissolved in pH 9.0 assay buffer (10 mL) to give a 10 mg/mL solution.
- WT-SBL (1.88 mg of dry powder, 73% w/w active protein) was dissolved in pH 5.8, 10 mM MES, 2 mM CaCl 2 "storage buffer” (500 ⁇ L) to give a 2.74 mg/mL solution of active WT-SBL. This solution was diluted four-fold with pH 5.8, 10 mM MES, 2 mM CaCl 2 "storage buffer” to give a 0.685 mg/mL solution.
- S156C-pyrazole and S166C-pyrazole were previously titrated with PMSF: their concentrations were 2.5 mg/mL and 3.62 mg/mL respectively. These stock solutions were diluted four-fold with pH 5.8, 10 mM MES, 2 mM CaCl 2 "storage buffer” to give 0.63mg/mL and 0.91 mg/mL solutions of S 156C-pyrazole and S 166C-pyrazole, respectively.
- PNPP solution p-Nitrophenyl phosphate solution
- WT-SBL 0.685 mg/mL
- S166C-pyrazole 0.91 mg/mL
- S156C-pyrazole 0.63 mg/mL.
- the tubes were kept on ice until aliquots had been withdrawn from each tube to establish initial HLADH and alkaline phosphatase activities. These activities were used to give "time zero" values for each tube (see below for assay protocols).
- the tubes were then incubated on a water bath at 35°C. Periodically, aliquots of reaction mixture were withdrawn from each eppendorf tube in order to assay HLADH and alkaline phosphatase activities.
- a portion of solution (65 ⁇ L) was withdrawn from an incubation vial and was then injected into a micro-cuvette containing pH 9.0 assay buffer (200 ⁇ L). The cuvette was incubated at 25°C for 2 minutes, and then cyclohexanol solution (30 ⁇ L) was added. The absorbance at 340 nm was then monitored for 120 s, and the O.D. change per second up to 0.2 absorbance units was recorded.
- Example 5 Synthesis of carbohydrate modified serine hydrolases The contamination of animal feed by certain lectins substantially reduces their nutritional value (Gatel (1994) Animal Feed Sci. Technl45: 317-348; Mogridge et al. (1996) J. Animal Sci. 74: 1897-1904; Pusztai et al. (1997) G. Brit. J. Nutrition 77, 933-945). In particular contamination of soy-based feeds by mannose-binding lectins prevents the effective use of crude feed without substantial purification.
- Example 6 Targeted Lectin Degradation Assay using Mannosylated-SBL. This example describes a highly effective lectin assay that has allowed us to start a screen of the ability of sugar-modified CMMs to degrade the lectin Concanavalin A in the manner shown schematically below ( Figure 14A, Figure 14B, and Figure 14C).
- S156C-sugar CMMs which contain surface exposed sugar groups were chosen initially.
- biotinylated lectin was incubated with glyco-CMM and compared with samples incubated with GG36-WT. To allow comparison, equal amounts of active enzyme were used. These samples were also incubated both with and without the decoy protein disulfide scrambled-RNaseA, in order to measure the selectivity of these enzymes for the lectin over the decoy.
- Figure 15A- Figure 15D A more detailed examination of Figure 15A- Figure 15D reveals that a) Released Biotin levels (indicating lectin degradation) are similar to each other. The presence of decoy reduces slightly both the level of GG36-WT and S156C- S-EtMan degradation. b) GG36-WT in the presence of decoy produces 18% more total protein after 210 min. than without. In contrast, S166C-S-EtMan in the presence of decoy produces only 7% more total protein - therefore the greater selectivity of S156C-S-EtMan reduces total protein absorption changes by 11%. c) Again, released Biotin levels (indicating lectin degradation) are similar to each other.
- Milli Q water blank (1 mL). d Calculated from difference between HABA/ Avidin drop in Abs. for sample and the drop in
- Milli Q water blank (1 mL). d Calculated from difference between HABA/ Avidin drop in Abs. for sample and the drop in
- the assay was performed as for method 1 except 100 ⁇ L aliquots of concanavalin replaced by 100 ⁇ L of Milli Q water. Results are shown in Table 27. Table 27. Assay of filtrate. Control without lectin.
- Biotinol (4) was elaborated, according to our established preparative procedure, to the target biotin-MTS via the co ⁇ esponding primary mesylate and bromide.
- the use of MsCl led to only a moderate yield of mesylate as a result of competing formation of primary chloride. Consequently, biotinol (4) was treated with mesylic anhydride in pyridine/DCM, then LiBr in refluxing acetone and finally NaSSO 2 Me in DMF to give target biotin-MTS 1 in 54% yield over 3 steps (37% overall yield from (+)-biotin (2)). Attempts to scale up this synthesis gave reduced yields.
- Biotin-MTS reagent 1 was used to prepare the biotinylated CMMs of N62C, L217C, S166C, and S156C mutants, by reaction at pH 9.5 following the standard protocol. In all cases the resulting enzymes are active after modification.
- the N62C-S-Biotin CMM had a slightly decreased k cat compared to SBL-WT and has the highest K M of all the biotin-CMMs, however it was still the most active of these biotinylated CMMs. Esterase Kinetics of the Biotin-CMMs
- GG36-WT 1940 180 0.54 0.07 3560 540
- the kca KM results for esterase activity follow the same trend compared to amidase kinetics.
- the S166C-S-Biotin CMM shows the smallest k cat IK M , which is about four times lower than for SBL-WT.
- the biotin-CMMs have an approximately four fold lower k cat compared to SBL-WT with the S 156C-S-Biotin CMM as the only exception.
- the k cat of the S 156C-S- Biotin CMM is about two fold lower than for SBL-WT and therefore two fold higher compared to the other biotin-CMMs.
- S156C-S-Biotin CMM is not the most active of all biotinylated CMMs since it has also the highest Rvalue.
- the k cat 'K M of S156C-S- Biotin CMM and L217C-S-Biotin CMM are very similar, about 3-fold lower than for SBL- WT, although k ca t and K M show big differences.
- the K M values of the biotin-CMMs were slightly higher for the S166C-S- Biotin CMM and the S156C-S-Biotin CMM compared to SBL-WT. Whereas, the values for the N62C-S-Biotin CMM and the L217C-S-Biotin CMM are about two times smaller compared to SBL-WT.
- the N62C-S-Biotin CMM has the lowest K M of all the biotinylated CMMs, and is 2.6 fold lower than SBL-WT. Although it also has the lowest k cat of the biotin-CMMs, it has the highest catalytic activity, which is still 1.8 fold lower then SBL-WT.
- biotinylated proteins will bind to avidin only when the biotin is separated from the surface of the macromolecule to which it is covalently linked by at least five methylene groups (Green (1970) Meth. Enzymol. 18 A: 418- 424). Furthermore, Wilchek et al. observed that proteolytic enzymes are not able to cleave avidin. Even when the proteases is biotinylated, avidin is not cleaved (Bayer et al. (1990) Biochemistry, 29: 11274-11279).
- the colorimetric method previously used to demonstrate lectin degradation with glycosylated CMMs was adapted, to assay the ability of the synthesized biotin-CMMs to target avidin.
- (+)-biotin should be better available to bind to avidin than the biotinylated side-chain of our CMMs we expected a smaller or, for the best case, the same HABA release for all biotin-CMMs compared to the biotin/WT mixture.
- Both SBL-WT and S156C-S-Biotin are able to hydrolyze avidin. Since the enzyme concentrations were calculated for equal catalytic activity with the standard amidase substrate suc-AAPF-pNA the hydrolysis values can be compared directly. Therefore we are able to demonstrate not only that avidin is hydrolyzed by SBL-proteases but is also more efficiently hydrolyzed by a biotinylated protease. S156C-S-Biotin produces 45% more protein fragments after 240 min than GG36-WT.
- Assay Buffer 20 mM Tris.HCl, 2 mM CaCl 2 , pH 8.6.
- b HABA/avidin reagent (Sigma) prepared with 10 mL of Milli-Q water.
- MES buffer 10 mM MES, 1 mM CaCl 2 , pH 5.8.
- Assay Buffer 20 mM Tris.HCl, 2 mM CaCl 2 , pH 8.6. b 5 mg/mL solution of avidin (Sigma) in Milli-Q water.
- the Avidin Hydrolysis Assay was also performed for N62C-S-Biotin and S166C-S-Biotin in addition to S156C-S-Biotin reported earlier. For comparison, we report the results for the S 156C CMM again.
- S156C-S-Biotin produces 72% more protein fragments after 240 min than SBL-WT.
- decoy protein [0.05 mg] the amount of total protein produced increases drastically for the WT enzyme (23% after 240 min) whereas the production of total protein does not change significantly for the biotin-CMM.
- N62C-S-Biotin provides nearly the same amount of protein fragments as
- the S166C CMM gives a 7% higher protein release compared to SBL-WT but is fairly unselective in the presence of a decoy protein [18% protein fragments compared to 23% for SBL-WT after 240 min]. Although this enzyme proved to be the second best of the biotin-CMMs in the "Avidin Targeting Assay", it is less selective than the N62C CMM with respect to the "Avidin Hydrolysis Assay". Presumably the biotin side chain buried in the Si pocket is available for avidin targeting but conformationally not very favorable for the effective and selective catalysis of avidin hydrolysis.
- Antibodies to biotin are commercially available as either free antibody or as an enzyme-conjugate. We chose an anti-biotin conjugated to alkaline phosphatase as our model target antibody. Using the standard Enzyme Linked Immuno- sorbent Assay (ELISA)-technique, we could demonstrate the ability of our CMMs to target the antibody. The experiment is outlined schematically in Figure 17.
- ELISA Enzyme Linked Immuno- sorbent Assay
- the phosphatase substrate [a solution of -nitrophenylphosphate disodium salt (PNPP) in glycine buffer pH 10.4] was added and the reaction was carried out at 4 °C. The release of -nitrophenolate was determined visually using a 96-well plate ( Figure 17).
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MXPA01010896A MXPA01010896A (en) | 1999-04-28 | 2000-04-21 | Specifically targeted catalytic antagonists and uses thereof. |
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JP2000613475A JP5180417B2 (en) | 1999-04-28 | 2000-04-21 | Specifically targeted catalytic antagonists and uses thereof |
CA2368213A CA2368213C (en) | 1999-04-28 | 2000-04-21 | Specifically targeted catalytic antagonists and uses thereof |
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US8637578B2 (en) | 2003-06-24 | 2014-01-28 | Isis Innovation Limited | Reagents and methods for the formation of disulfide bonds and the glycosylation of proteins |
US9856283B2 (en) * | 2013-03-14 | 2018-01-02 | The Regents Of The University Of California | Thiosaccharide mucolytic agents |
NL2028505B1 (en) * | 2021-06-21 | 2022-12-29 | Pharmacytics B V | Sugar thiol compounds and methods of producing the same |
US12187762B2 (en) | 2018-09-10 | 2025-01-07 | The Regents Of The University Of California | Dithiolsaccharide mucolytic agents and uses thereof |
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US20030003114A1 (en) * | 2001-03-22 | 2003-01-02 | Pan Xing Qing | Enzyme-based anti-cancer compositions and methods |
GB0404731D0 (en) * | 2004-03-03 | 2004-04-07 | Indp Administrative Inst Nims | Method and products for the selective degradation of proteins |
US8034921B2 (en) * | 2006-11-21 | 2011-10-11 | Alnylam Pharmaceuticals, Inc. | IRNA agents targeting CCR5 expressing cells and uses thereof |
US7760357B2 (en) * | 2008-06-18 | 2010-07-20 | David Wagner | Tachyonized material test method |
GB2552195A (en) * | 2016-07-13 | 2018-01-17 | Univ Oxford Innovation Ltd | Interferometric scattering microscopy |
GB2588378A (en) | 2019-10-10 | 2021-04-28 | Refeyn Ltd | Methods and apparatus for optimised interferometric scattering microscopy |
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
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US8637578B2 (en) | 2003-06-24 | 2014-01-28 | Isis Innovation Limited | Reagents and methods for the formation of disulfide bonds and the glycosylation of proteins |
US9856283B2 (en) * | 2013-03-14 | 2018-01-02 | The Regents Of The University Of California | Thiosaccharide mucolytic agents |
US10526359B2 (en) | 2013-03-14 | 2020-01-07 | The Regents Of The University Of California | Thiosaccharide mucolytic agents |
AU2019200171B2 (en) * | 2013-03-14 | 2020-05-07 | The Regents Of The University Of California | Thiosaccharide mucolytic agents |
US11021506B2 (en) | 2013-03-14 | 2021-06-01 | The Regents Of The University Of California | Thiosaccharide mucolytic agents |
AU2020213367B2 (en) * | 2013-03-14 | 2022-10-06 | The Regents Of The University Of California | Thiosaccharide mucolytic agents |
US12187762B2 (en) | 2018-09-10 | 2025-01-07 | The Regents Of The University Of California | Dithiolsaccharide mucolytic agents and uses thereof |
NL2028505B1 (en) * | 2021-06-21 | 2022-12-29 | Pharmacytics B V | Sugar thiol compounds and methods of producing the same |
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US20040170618A1 (en) | 2004-09-02 |
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